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| United States Patent |
6,171,589
|
|
Browning
, et al.
|
January 9, 2001
|
Mycoplasma recombinant polypeptides and vaccines
Abstract
The present invention relates generally to immunogens and their use in
vaccine preparations. More particularly, the present invention is directed
to a peptide or polypeptide or a derivative, homologue or analogue thereof
which corresponds to, mimics, or cross-reacts with, B-cell or T-cell
epitopes on surface polypeptides encoded by Mycoplasma pneumoniae or M.
genitalium. The immunogens of the present invention are particularly
useful in vaccine preparations for the prophylactic and therapeutic
treatment of individuals against infections by Mycoplasma ssp. The present
invention further provides diagnostic reagents for the detection of
Mycoplasma ssp. in biological samples derived from individuals suspected
of being infected therewith.
| Inventors:
|
Browning; Glenn Francis (Parkville, AU);
Duffy; Michael Francis (Parkville, AU);
Whithear; Kevin George (Parkville, AU);
Walker; Ian Douglas (Parkville, AU)
|
| Assignee:
|
The University of Melbourne (Parkville, AU)
|
| Appl. No.:
|
091117 |
| Filed:
|
September 2, 1998 |
| PCT Filed:
|
December 13, 1996
|
| PCT NO:
|
PCT/AU96/00803
|
| 371 Date:
|
September 2, 1998
|
| 102(e) Date:
|
September 2, 1998
|
| PCT PUB.NO.:
|
WO97/21727 |
| PCT PUB. Date:
|
June 19, 1997 |
Foreign Application Priority Data
| Current U.S. Class: |
424/184.1; 424/190.1; 530/300; 530/350; 530/387.1 |
| Intern'l Class: |
A61K 039/06; A61K 039/38; A61K 039/02; A61K 038/00; C07K 001/00 |
| Field of Search: |
424/184.1,190.1
530/350,300,387.1
|
References Cited
Other References
Lin Juey-Shin, An antigenic analysis for membranes of Mycoplasma hominis by
cross absorption. Journal of General Microbiology, 116, 187-193, 1980.*
Liepmann et al, Use of the Mycoplasma hominis 102-116 kD proteins as
antigen in an enzyme-linked immunosorbent assay. Microbios, 65(262) 7-13,
1991.*
Krause, DC and Robinson, DT, Mycoplasmas Molecular Biology and
Pathogenesis, Mycoplasmas which infect humans, Chapter 25, pp. 417-444,
1992.*
Fraser, C.M. et al., "The minimal Gene Complement of Mycoplasma
genitalium," (1995) Science 270:397-403.
Himmelreich, R. et al., "Complete sequence analysis of the genome of the
bacterium Mycpolasma pneumonia," (1996) Nucleic Acids Research 24(22):
4420-4449.
|
Primary Examiner: Minnifield; Nita
Assistant Examiner: Baskar; Padma
Attorney, Agent or Firm: Greenlee Winner and Sullivan PC
Claims
What is claimed is:
1. An isolated or recombinant immunogenic polypeptide of a Mycoplasma ssp.
having a molecular weight selected from the group consisting of: (i) a
predicted molecular weight as determined from the amino acid sequence of
said polypeptide of approximately 16 kDa or 116 kDa; and (ii) a molecular
weight of 110 kDa as determined using SDS/PAGE and wherein said
polypeptide comprises an amino acid sequence selected from the group
consisting of:
(i) the amino acid sequence set forth in SEQ ID NO:1;
(ii) the amino acid sequence set forth in SEQ ID NO:2;
(iii) the amino acid sequence set forth in SEQ ID NO:4;
(iv) the amino acid sequence set forth in SEQ ID NO:5;
(v) an amino acid sequence having at least 70% sequence identity to any one
of (i) to (iv), wherein said polypeptide is immunogenic; and
(vi) an immunogenic fragment of any one of (i) to (iv).
2. The isolated or recombinant immunogenic polypeptide according to claim 1
wherein said polypeptide is isolated from Mycoplasma pneumoniae or wherein
said polypeptide is isolated from a recombinant bacterial cell expressing
said polypeptide.
3. The isolated or recombinant immunogenic polypeptide according to claim 2
wherein said polypeptide is a surface polypeptide which has adhesion
properties.
4. The isolated or recombinant immunogenic polypeptide according to claim 1
wherein the polypeptide has an amino acid of an immunogenic fragment
comprising an amino acid sequence selected from the group consisting of:
(i) amino acid residues 9 to 473 of SEQ ID NO:2;
(ii) amino acid residues 467-709 of SEQ ID NO:2;
(iii) amino acid residues 709 to 850 of SEQ ID NO:2;
(iv) amino acid residues 846 to 896 of SEQ ID NO:2;
(v) amino acid residues 887 to 962 of SEQ ID NO:2; and
(vi) amino acid residues 969 to 1029 of SEQ ID NO:2.
5. The isolated or recombinant immunogenic polypeptide according to claim 4
wherein the polypeptide has an amino acid sequence of an immunogenic
fragment comprising an amino acid residues 9 to 473 of SEQ ID NO:2 or a
fragment thereof comprising a B or T cell epitope.
6. The isolated or recombinant immunogenic polypeptide according to claim 1
wherein said polypeptide is encoded by an isolated DNA molecule selected
from the group consisting of:
(i) a DNA molecule comprising a sequence of nucleotides of SEQ ID NO:3;
(ii) a DNA molecule comprising a nucleotide sequence having at least 70%
identity to SEQ ID NO:3;
(iii) a DNA molecule comprising a nucleotide sequence that is capable of
hybridizing to SEQ ID NO:3; and
(iv) a fragment of (i) that encodes an immunogenic fragment of said
immunogenic polypeptide.
7. The isolated or recombinant immunogenic polypeptide according to claim 1
derived from Mycoplasma ssp. selected from the group consisting of M.
penetrans, M. iowae, M. gallisepticum, M. genitalium, M. imitans, M.
muris, M. urealyticum and M. pirum.
8. The isolated or recombinant immunogenic polypeptide according to claim 7
derived from M. genitalium.
9. The isolated or recombinant immunogenic polypeptide according to claim 1
wherein said polypeptide is a surface polypeptide.
10. The isolated or recombinant immunogenic polypeptide according to claim
1 when expressed in a virus particle, prokaryotic cell or eukaryotic cell.
11. The isolated or recombinant immunogenic polypeptide according to claim
10 wherein the prokaryotic cell is a bacterial cell.
12. The isolated or recombinant immunogenic polypeptide according to claim
11 wherein the bacterial cell is an Escherichia coli cell or a Mycoplasma
ssp. cell.
13. The isolated or recombinant immunogenic polypeptide according to claim
10 wherein said polypeptide is expressed as a fusion polypeptide.
14. The isolated or recombinant immunogenic polypeptide according to claim
13 wherein the fusion polypeptide is a fusion with
glutathione-s-transferase.
15. A vaccine composition for the therapeutic or prophylactic treatment of
a mammalian subject against infection by a Mycoplasma ssp., said
composition comprising the isolated or recombinant immunogenic polypeptide
according to claim 1 in an amount sufficient to mediate an immune response
when administered to said mammal and a pharmaceutically acceptable carrier
or diluent.
16. The vaccine composition according to claim 15 wherein the mammal is a
human.
17. The vaccine composition according to claim 15 wherein the means of
administration is injection or ingestion.
18. The vaccine composition according to claim 15 wherein the Mycoplasma
ssp. is M. pneumoniae.
19. The vaccine composition according to claim 15 wherein said vaccine
composition is capable of inducing humoral immunity against said
Mycoplasma ssp.
20. The vaccine composition according to claim 19 wherein said vaccine
composition further prevents the onset, development or progression of
symptoms associated with M. pneumoniae infection with administered to said
mammal.
21. The vaccine composition according to claim 20 wherein the symptoms
associated with M. pneumoniae infection are selected from the list
comprising atypical pneumoniae, lung lesions and inflammatory reactions of
the respiratory tract or central nervous system.
22. The vaccine composition according to claim 15 further comprising an
adjuvant.
23. An isolated nucleic acid molecule wherein said nucleic acid molecule
encodes an isolated or recombinant immunogenic polypeptide which comprises
an amino acid sequence having at least 70% identity to any of SEQ ID
NOs:1, 2, 4 or 5.
24. A genetic construct comprising the isolated nucleic acid molecule
according to claim 23 operably linked to a promoter sequence which is
capable of regulating expression of said nucleic acid molecule in a virus
particle, prokaryotic cell or eukaryotic cell.
25. The genetic construct according to claim 24 when used to produce a
recombinant polypeptide which comprises an amino acid sequence having at
least 70% identity to any one of SEQ ID NOs:1, 2, 4 or 5.
26. The genetic construct according to claim 25 wherein the recombinant
polypeptide is a polypeptide immunogen.
27. An isolated or recombinant immunogenic polypeptide of Mycoplasma ssp.
having a molecular weight selected from the group consisting of: (i) a
predicted molecular weight as determined from the amino acid sequence of
said polypeptide of approximately 16 kDa or 116 kDa; and (ii) a molecular
weight of 110 kDa as determined using SDS/PAGE wherein said isolated or
recombinant polypeptide is detected by a process comprising:
(i) hybridizing nucleic acid of Mycoplasma spp. to a probe or primer
comprising the nucleotide sequence set forth in SEQ ID NO:3 under at least
low stringency hybridization conditions and isolating the hybridized
nucleic acid;
(ii) expressing the hybridized nucleic acid in a cell, tissue, organ or
organism for a time and under conditions sufficient for transcription and
translation of said nucleic acid to occur; and
(iii) detecting the expressed protein.
28. An isolated or recombinant immunogenic polypeptide of Mycoplasma ssp.
having a molecular weight selected from the group consisting of: (i) a
predicted molecular weight as determined from the amino acid sequence of
said polypeptide of approximately 16 kDa or 116 kDa; and (ii) a molecular
weight of 110 kDa as determined using SDS/PAGE wherein said isolated or
recombinant polypeptide is detected by a process comprising contacting
protein of said Mycoplasma with an antibody molecule that binds
specifically to the isolated or recombinant protein of claim 1.
Description
The present invention relates generally to peptides and polypeptides and
their use in vaccine preparations. More particularly, the present
invention is directed to a peptide or polypeptide or a derivative,
homologue or analogue thereof which corresponds to, mimics, or
cross-reacts with, B-cell or T-cell epitopes on polypeptides encoded by
Mycoplasma pneumoniae and M. genitalium. Vaccine preparations comprising
the peptides or polypeptides of the present invention are useful in
protecting individuals against infections by species of the genus
Mycoplasma.
Throughout this specification and the claims which follow, unless the
context requires otherwise, the word "comprise", or variations such as
"comprises" or "comprising", will be understood to imply the inclusion of
a stated integer or group of integers but not the exclusion of any other
integer or group of integers.
Bibliographic details of the publications referred to by author in this
specification are collected at the end of the description.
Sequence identity numbers (SEQ ID Nos.) for the nucleotide and amino acid
sequences referred to in the specification are defined after the
bibliography.
The micro-organisms Mycoplasma pneumoniae is a pathogen of humans that
typically colonises the upper respiratory tract. Mycoplasma pneumoniae
moves along the cilia of the respiratory epithelium until in close
association with the host cell to which it adheres. It disrupts the
protein ciliary necklace at the base of the host cell cilia causing
ciliostasis.
The threat the M. pneumoniae poses to children and sensitized adults is
considerable. Mycoplasma pneumoniae is the primary cause of atypical
pneumonia in young adults and children, although infected patients often
present with symptoms similar to a persistent influenza infection. A study
performed in the U.K. (Granstrom et al, 1994) has attributed 18% of the
total number of cases of acquired pneumonia in the community, to M.
pneumoniae infection. Periodically, M. pneumoniae is present in epidemic
proportions in human communities. Previous exposure to the pathogen can
result in hypersensitivity reactions upon reinfection (Cimolai et al,
1992).
Although only one in one thousand cases of M. pneumoniae infection result
in pathology of the central nervous system (CNS), infection is associated
with 5-10% of cases of neurological syndromes (Koskiniemi, 1993). Typical
CNS manifestations associated with infection include encephalitis,
meningitis and myelitis. Additional complications associated with M.
pneumoniae infection include the presence of cold agglutinins and
arthropathy (Cimolai et al, 1989). A report of Zagami et al. (1994)
suggests that the neurological pathology of M. pneumoniae associated
encephalitis results from a cell mediated response to shared M. pneumoniae
antigens, or a local inflammatory response of the CNS due to the presence
of M. pneumoniae in the CNS.
However, notwithstanding the endemic and serious nature of M. pneumoniae
related disease, no means is available for the accurate and rapid
diagnosis of M. pneumoniae infection, or for the prophylaxis of
individuals exposed to infection. Accordingly, there is a clear need to
develop agents useful in the diagnosis and prophylaxis of infection by
mycoplasmas, in particular M. pneumoniae.
Previous attempts to develop a suitable peptide vaccine against M.
pneumoniae have been largely unsuccessful. For example, although hamsters
immunised with M. pneumoniae may develop antibodies against the P1 protein
of M. pneumoniae, the results, in terms of the degree of humoral immunity
conferred by such immunisation, have been inconsistent and unpredictable
(Yayoshi et al, 1992). In particular, hamsters inoculated with the P24-SII
live vaccine, which contains the P1 protein but not the 85 kDa protein,
were not protected against infection. The P24-SI live vaccine, containing
the P1 protein and the 85 kDa protein, gave 50% protection against
infection. The FH-P24 live vaccine, also containing both the P1 protein
and the 85 kDa protein, gave 90% protection. The P24-SI and P24-SII
vaccines are nitroso guanidine non-hemolysing mutants of FH-P24.
Furthermore, no success has been obtained using the 43 kDa M. pneumoniae
protein, which appears to enhance the severity of infection by this
pathogen (Cimolai et al, 1992).
In work leading up to the present invention, the inventors sought to
develop better and more effective vaccines and diagnostic agents for M.
pneumoniae, by cloning. M. pneumoniae genes encoding highly immunogenic
polypeptides and homologues and derivatives thereof. The recombinant
polypeptides and derivatives, homologues or analogues thereof, provide the
means to develop a range of diagnostic and prophylactic agents for
Mycoplasma infection which were hitherto not available.
Accordingly, one aspect of the present invention is directed to an isolated
or recombinant polypeptide or a derivative, homologue or analogue thereof
wherein said polypeptide is obtainable from a species of Mycoplasma.
In one embodiment of the present invention, there is provided an isolated
or recombinant polypeptide or derivative, homologue or analogue thereof
wherein said polypeptide is obtainable from a species of Mycoplasma and
has a predicted molecular weight of approximately 16 kDa.
Preferably, the isolated or recombinant polypeptide of the present
invention or a derivative, homologue or analogue thereof is further
obtainable from Mycoplasma pneumoniae or M. genitalium and M. genitalium.
More preferably, said polypeptide further comprises an amino acid sequence
substantially the same as the amino acid sequence set forth in SEQ ID NO:1
or SEQ ID NO:4, or is at least 70% similar to all or a part thereof.
In one particularly preferred embodiment, the isolated or recombinant
polypeptide or a derivative, homologue or analogue thereof is
characterised by any one or more of the following properties:
(I) it is obtainable from Mycoplasma pneumoniae;
(ii) it has a predicted molecular weight of approximately 16 kDa; or
(iii) it comprises an amino acid sequence substantially as set forth in SEQ
ID NO:1 or having at least 70% similarity to all or a part thereof.
Alternatively or in addition, the isolated or recombinant polypeptide or a
derivative, homologue or analogue thereof is characterised by any one of
the following properties:
(I) it is obtainable from Mycoplasma genitalium;
(ii) it has a predicted molecular weight of approximately 16 kDa; or
(iii) it comprises an amino acid sequence substantially as set forth in SEQ
ID NO:4 or having at least 70% similarity to all or a part thereof.
The homologous amino acid sequences set forth in SEQ ID Nos:1 and 4 are
only 37.3% identical overall, as described in Example 21. Accordingly, the
present invention further extends to any isolated Mycoplasma polypeptide
which has properties of a surface polypeptide and is at least 35%
identical to SEQ ID NO:1 or SEQ ID NO:4.
In an alternative embodiment of the present invention, there is provided an
isolated polypeptide, or a derivative, homologue or analogue thereof
wherein said polypeptide is obtainable from a species of Mycoplasma and
wherein said polypeptide in its native form is a surface polypeptide which
has adhesion properties.
Preferably, one embodiment is directed to an isolated polypeptide, or a
derivative, homologue or analogue thereof wherein said polypeptide is
obtainable from Mycoplasma pneumoniae, has a molecular weight of
approximately 110 kDa determined by SDS/PAGE, or a predicted molecular
weight of approximately 116 kDa and in its native form is a surface
polypeptide which has adhesion properties.
For the present purposes, it will be understood that reference to the
molecular weight of the subject polypeptide does not necessarily limit the
invention, but is included especially for the purposes of nomenclature.
Those skilled in the art are aware of the degree of precision associated
with molecular weight estimates in respect of protein or polypeptide
molecules and the fact that such estimates vary considerably depending
upon the means employed to obtain them. For example, the subject
polypeptide having derived molecular weight of 116 kDa may electrophorese
on SDS/polyacrylamide gels such that it has an estimated molecular weight
of only 110 kDa. Accordingly, reference herein to the term "116 kDa
polypeptide" or "110 kDa polypeptide" are not to be taken as mutually
exclusive definitions.
Even more particularly, this embodiment of the present invention is
directed to an isolated polypeptide or derivative, homologue or analogue
thereof characterised by the following properties:
(I) it is obtainable from Mycoplasma pneumoniae;
(ii) it has a molecular weight of approximately 110 kDa as determined by
SDS/PAGE, or a predicted molecular weight of approximately 116 kDa;
(iii) it is a surface polypeptide in its native form;
(iv) it has adhesion properties in its native form; or
(v) it comprises an amino acid sequence substantially as set forth in SEQ
ID NO:2 or having at least 70% similarity to all or a part thereof.
Alternatively, this embodiment is directed to an isolated polypeptide, or a
derivative, homologue or analogue thereof wherein said polypeptide is
obtainable from Mycoplasma genitalium.
Accordingly, this alternative embodiment is directed to an isolated
polypeptide or derivative, homologue or analogue thereof characterised by
any of the following properties:
(I) it is obtainable from Mycoplasma genitalium;
(ii) it is at least 50% identical to the amino acid sequence set forth in
SEQ ID NO:2;
(iii) it has the structural properties of a surface polypeptide; or
(iv) it comprises an amino acid sequence substantially as set forth in SEQ
ID NO:5 or having at least 70% similarity to all or a part thereof.
The present invention extends to both isolated non-recombinant
polypeptides, recombinant polypeptides and isolated recombinant
polypeptides of Mycoplasma described in any of the foregoing embodiments.
In particular, the present invention extends to isolated non-recombinant
polypeptides, recombinant polypeptides and isolated recombinant
polypeptides comprising a sequence of amino acids substantially as set
fort in SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID NO:5 or having
at least 70% similarity to all or a part thereof.
According to the foregoing embodiments, when the polypeptide of the present
invention is a recombinant polypeptide, it may be produced in and, if
desirable isolated from, any virus particle or cell. As will be known to
those skilled in the relevant art, a cell for production of a recombinant
polypeptide is selected on the basis of several parameters including the
genetic constructs used to express the polypeptide under consideration,
stability and activity of said polypeptide. It will also be known to those
skilled in the art, that the stability or activity of a recombinant
polypeptide may be determined, at least in part, by post-translational
modifications to the polypeptide, for example glycosylation, acylation or
alkylation reactions, amongst others, which may vary between cell lines
used to produce the recombinant polypeptide.
The present invention extends further to a recombinant polypeptide
according to any of the foregoing embodiments or a derivative, homologue
or analogue thereof, wherein said polypeptide is produced in any virus
particle or a prokaryotic or eukaryotic cell or a culture thereof.
In a preferred embodiment, the present invention extends to a recombinant
polypeptide according to any of the foregoing embodiments or a derivative,
homologue or analogue thereof, wherein said polypeptide is produced in a
bacterial cell or culture thereof belonging to the genus Mycoplasma, in
particular a cell of M. pneumoniae or M. genitalium or a culture thereof
or an Escherichia coli cell.
The term "polypeptide" as used herein shall be taken to refer to any
polymer consisting of amino acids linked by covalent bonds and includes
within its scope full-length proteins and parts or fragments thereof, for
example oligopeptides and short peptide sequences consisting of at least
two amino acid residues. Also included within the scope of the definition
of a "polypeptide" are amino acid sequence variants, containing amino acid
substitutions, deletions, or insertions which do not alter the essential
properties of said polypeptide, for example its immunogenicity or
effectiveness as a peptide vaccine against Mycoplasma ssp, amongst others.
Accordingly, a polypeptide may be isolated from a source in nature, or
chemically synthesized. Furthermore, a polypeptide may be derived from a
full-length protein by chemical or enzymatic cleavage, using reagents such
as CNBr, trypsin, or chymotrypsin, amongst others.
The term "recombinant polypeptide" as used herein shall be taken to refer
to a polypeptide which is produced in a virus particle or a cell by the
expression therein of a genetic sequence encoding said polypeptide under
the control of a suitable promoter, wherein a genetic manipulation has
been performed in order to achieve said expression. Genetic manipulations
will be known to those skilled in the art and include, but are not limited
to nucleic acid isolation, digestion, ligation, amplification,
hybridisation or sequencing.
The term "surface polypeptide" or similar term as used herein shall be
taken in its broadest context to refer to a polypeptide which is localised
on, or intrinsically or extrinsically associated with, the surface layer
of Mycoplasma spp. and in particular M. pneumoniae or M. genitalium. A
surface polypeptide, or at least an epitope thereof, is accessible to
recognition by the immune system of the host organism without lysis of the
infecting pathogen.
The term "adhesion properties" as used herein shall be taken to refer to a
functional characteristic of a polypeptide which facilitates the
association, adherence or attachment of a micro-organism to a cell of a
host organism during the infectious phase. In the present context,
adhesion properties of the micro-organism usually render M. pneumoniae
capable of binding host cells of the upper respiratory tract.
The term "native form" or "native state" or similar term as used herein
with reference to a characteristic of a polypeptide, shall be taken as a
reference to the inherent properties of a non-recombinant polypeptide when
it is present in the cell from which it originates, such as a mycoplasma
cell, in particular a M. pneumoniae cell. For example, the M. pneumoniae
polypeptide set forth in SEQ ID NO:2 has a predicted molecular weight of
116 kDa and is said to be in its native state when it is in a M.
pneumoniae cell and no genetic manipulations have been performed upon it.
Accordingly, said polypeptide when present in a M. pneumoniae cell, has
the inherent properties of being localised to the cell surface and is an
adhesion polypeptide.
The term "predicted molecular weight" as used herein in relation to a
polypeptide refers to a molecular weight which is determined by a
summation of the molecular weights of individual chemical elements or
atoms comprised therein.
In a more particularly preferred embodiment, the present invention extends
to a recombinant polypeptide or a derivative, homologue or analogue
thereof produced in a virus particle, prokaryotic or eukaryotic cell or a
virus or cell culture thereof, wherein said recombinant polypeptide has an
amino acid sequence which is substantially the same as the amino acid
sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 or a part thereof.
It is understood in the art that although parts or fragments of a
full-length protein may not possess all of the properties of the
full-length protein, they are at least useful as diagnostic reagents, or
as immunogens in the production of immunoreactive molecules or vaccine
preparations. Accordingly, the present invention extends to parts or
fragments of an isolated polypeptide defined according to the embodiments
herein.
In a particularly preferred embodiment, the present invention extends to a
part or fragment of the isolated or recombinant form of a polypeptide set
forth in SEQ ID NO:1 or SEQ ID NO:2
In a more particularly preferred embodiment of the invention, there is
provided a fragment or derivative of the amino acid sequence of the forth
in SEQ ID NO:2 comprising amino acid residues 9 to 473, 467 to 709, 709 to
850, 846 to 896, 887 to 962 or 969 to 1029 of said amino acid sequence or
a homologue, analogue or derivative thereof. The specific fragments or
derivatives of SEQ ID NO: 2 to which this embodiment of the invention
relates are exemplified in Table 5 which is incorporated herein.
Even more particularly preferred, the fragment or derivative of SEQ ID NO:2
is highly immunogenic or antigenic, preferably when compared to the
full-length amino acid sequence. Those skilled in the art will be aware
that such peptide derivatives or fragments are useful as immunogens which
comprise a B cell or T cell epitope. Particularly suitable for this
purpose is a fragment or derivative of SEQ ID NO: 2 comprising amino acid
residues 9 to 473 of SEQ ID NO: 2 or a homologue, analogue or derivative
thereof.
The polypeptide, or a derivative, homologue or analogue thereof of the
present invention is particularly useful as a polypeptide component in a
vaccine composition which is designed to protect an individual against
infection by a species of Mycoplasma, in particular to protect an
individual against infection by Mycoplasma pneumoniae or M. genitalium.
The essential feature of said polypeptide, or derivative, homologue or
analogue thereof for the present purpose is whether it is "immunogenic",
defined hereinafter as the ability of said polypeptide, or a derivative,
homologue or analogue thereof, to elicit B cell and/or T cell responses in
the host, as part of the immunization process. It is understood by a
person skilled in the art that not all parts of a polypeptide or
derivative, homologue or analogue thereof are equally immunogenic. In
fact, a polypeptide or a derivative, homologue or analogue thereof may be
comprised of several different overlapping, or non-overlapping regions,
which, in isolation, or in combination, are highly immunogenic. Said
highly immunogenic region, or a derivative, homologue or analogue thereof,
is hereinafter referred to as a "B cell and/or T cell epitope".
Furthermore, a B cell or T cell epitope of a polypeptide or a derivative,
homologue or analogue thereof may comprise any one or more of the
following:
(I) the primary amino acid sequence of said region, known in the art as a
continuous non-conformational epitope;
(ii) the secondary structure which said region adopts, known in the art as
a continuous conformational epitope;
(iii) the tertiary structure which said region adopts in contact with
another region of the same polypeptide molecule, known in the art as a
discontinuous conformational epitope; or
(iv) the quaternary structure which said region adopts in contact with a
region of another polypeptide molecule, known in the art as a
discontinuous conformational epitope.
Accordingly, immunogenic polypeptides or derivatives, homologues or
analogues thereof comprising the same, or substantially the same primary
amino acid sequence are hereinafter defined as "immunogens which comprise
a B cell or T cell epitope", or similar term.
Immunogenic polypeptides or derivatives, homologues, or analogues thereof
comprising different primary amino acid sequences may comprise
immunologically identical immunogens, because they possess conformational
B cell or T cell epitopes that are recognised by the immune system of a
host species to be identical. Such immunogenic polypeptides or
derivatives, homologues or analogues thereof are hereinafter defined as
"immunogens which mimic or cross-react with a B cell or T cell epitope",
or similar term.
Accordingly, the present invention extends to an immunogen which comprsies,
mimics, or cross-reacts with a B-cell or T-cell epitope of an isolated or
recombinant polypeptide according to any of the foregoing embodiments or a
derivative, homologue or analogue thereof. In a particularly preferred
embodiment, the present invention provides an immunogen which comprises,
mimics, or cross-reacts with a B-cell or T-cell epitope of an isolated or
recombinant polypeptide which in its native form is obtainable from a
species of Mycoplasma such as, but not limited to M. pneumoniae and has a
predicted molecular weight of approximately 16 kDa or a homologue,
analogue or derivative thereof. According to this embodiment, when the
polypeptide has a predicted molecular weight of approximately 116 kDa it
is also preferred that said polypeptide in its native form is a surface
polypeptide with adhesion properties.
In a particularly preferred embodiment of the invention, there is provided
an immunogen comprising amino acid resides 9 to 473, 467 to 709, 709 to
850, 846 to 896, 887 to 962 or 969 to 1029 of SEQ ID NO: 2 or a homologue,
analogue or derivative thereof. More particularly, the preferred
immunogen, according to this embodiment of the invention, comprises amino
acid residues 9 to 473 of SEQ ID NO: 2 or a homologue, analogue or
derivative thereof.
In an alternative embodiment, the present invention extends to an immunogen
which comprises, mimics, or cross-reacts with a B-cell or T-cell epitope
of a polypeptide according to any of the foregoing embodiments or a
derivative, homologue or analogue thereof, wherein said polypeptide
comprises a sequence of amino acids which is at least about 40% similar to
the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2, more
preferably at least about 60% similar, still more preferably at least
about 80% similar and even still more preferably, at least about 99%
similar to the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID
NO:2.
In the present context, "homologues" of a polypeptide refer to those
polypeptides, enzymes or proteins which have similar properties as a
polypeptide of the present invention, for example surface protein adhesion
properties or immunogenic properties as described supra, notwithstanding
any amino acid substitutions, additions or deletions thereto.
Furthermore, amino acids may be replaced by other amino acids having
similar properties, for example hydrophobicity, hydrophilicity,
hydrophobic moment, antigenicity, propensity to form a break
.alpha.-helical structures or .beta.-sheet structures, and so on.
The present invention clearly extends to such amino acid variants, provided
that such molecules still function as B cell or T-cell epitopes capable of
mediating an immune response or functioning as a surface polypeptide.
Preferably, a homologue will still function as a peptide immunogen which
mimics or cross-reacts to B-cell or T-cell epitopes of a Mycoplasma spp.
polypeptide of the present invention.
Furthermore, a homologue may be isolated or derived from the same or
another Mycoplasma species. Preferred sources of homologues of a
Mycoplasma pneumoniae polypeptide according to the present invention are
M. genitalium, M. penetrans, M. iowae, M. gallisepticum, M. imitans, M.
muris, M. urealyticum or M. pirum, amongst others. In a particularly
preferred embodiment of the invention, homologues of the M. pneumoniae 16
kDa and 116 kDa polypeptide are isolated from M. genitalium. The amino
acid sequence of the M. genitalium homologue of the 16 kDa polypeptide is
set forth in SEQ ID NO:4. The amino acid sequence of the M. genitalium
homologue of the 116 kDa polypeptide is set forth in SEQ ID NO:5.
Substitutions encompass amino acid alterations in which an amino acid is
replaced with a different naturally-occurring or a non-conventional amino
acid residue. Such substitutions may be classified as "conservative", in
which case an amino acid residue contained in a repressor polypeptide is
replaced with another naturally-occurring amino acid of similar character,
for example Gly{character pullout}Ala, Val{character pullout}Ile{character
pullout}Leu, Asp{character pullout}Glu, Lys{character pullout}Arg,
Asn{character pullout}Gln or Phe{character pullout}Trp{character
pullout}Tyr.
Substitutions encompassed by the present invention may also be
"non-conservative", in which an amino acid residue is substituted with an
amino acid having different properties, such as a naturally-occurring
amino acid from a different group (eg. substituted a charged or
hydrophobic amino acid with alanine), or alternatively, in which a
naturally-occurring amino acid is substituted with a non-conventional
amino acid.
Amino acid substitutions are typically of single residues, but may be
clustered depending upon functional constraints placed upon the
polypeptide; insertions will usually be of the order of about 1-10 amino
acid residues; and deletions will range from about 1-20 residues. Amino
acid alterations to the peptides contemplated herein include insertions
such as amino acid and/or carboxyl terminal fusions as well as
intra-sequence insertions of single or multiple amino acids. Generally,
insertions within the amino acid sequence will be smaller than amino or
carboxyl terminal fusions, of the order of about 1 to 4 residues.
Insertional amino acid sequence variants are those in which one or more
amino acid residues are introduced into a predetermined site in the
protein. Deletional variants are characterised by the removal of one or
more amino acids from the sequence. Substitutional variants are those in
which at least one residue in the sequence has been removed and a
different residue inserted in its place. Such substitutions may be made in
accordance with Table 1.
The amino acid variants referred to in Table 1 may readily be made using
peptide synthetic techniques well known in the art, such as solid phase
peptide synthesis and the like, or by recombinant DNA manipulations.
Techniques for making substitution mutations at predetermined sites in DNA
having known sequence are well known, for example through M13 mutagenesis.
The manipulation of DNA sequences to produce variant proteins which
manifest as substitutional, insertional or deletional variants are well
known in the art.
TABLE 1
Three-letter One-letter
Amino Acid Abbreviation Symbol
Alanine Ala A
Arginine Arg R
Asparagine Asn N
Aspartic acid Asp D
Cysteine Cys C
Glutamine Gln Q
Glutamic acid Glu E
Glycine Gly G
Histidine His H
Isoleucine Ile I
Leucine Leu L
Lysine Lys K
Methionine Met M
Phenylalanine Phe F
Proline Pro P
Serine Ser S
Threonine Thr T
Tryptophan Trp W
Tyrosine Tyr Y
Valine Val V
"Analogues" encompass polypeptides which are functionally equivalent or at
least have similar properties as a polypeptide of the present invention,
notwithstanding the occurrence of any non-naturally occurring or modified
amino acid residues therein. Non-naturally occurring amino acid residues
contemplated in an analogue of the present invention are set forth in
Table 2.
TABLE 2
Non-conventional Non-conventional
amino acid Code amino acid Code
.alpha.-aminobutyric acid Abu L-N-methylalanine Nmala
.alpha.-amino-.alpha.-methylbutyrate Mgabu L-N-methylarginine Nmarg
aminocyclopropane- Cpro L-N-methyl- Nmasn
carboxylate asparagine
L-N-methylaspartic Nmasp
acid
aminoisobutyric acid Aib L-N-methylcysteine Nmcys
aminonorbornyl- Norb L-N-methylgluta- Nmgln
carboxylate mine
L-N-methylglutamic Nmglu
acid
cyclohexylalanine Chexa L-N-methylhistidine Nmhis
cyclopentylalanine Cpen L-N-methylisol- Nmile
leucine
D-alanine Dal L-N-methylleucine Nmleu
D-arginine Darg L-N-methyllysine Nmlys
D-aspartic acid Dasp L-N-methylmethio- Nmmet
nine
D-cysteine Dcys LN-methylnor- Nmnle
leucine
D-glutamine Dgln L-N-methylnorva- Nmnva
line
D-glutamic acid Dglu L-N-methylornithine Nmorn
D-histidine Dhis L-N-methylphenyla- Nmphe
laline
D-isoleucine Dile L-N-methylproline Nmpro
D-leucine Dleu L-N-methylserine Nmser
D-lysine Dlys L-N-methylthreo- Nmthr
nine
D-methionine Dmet L-N-methyltrypto- Nmtrp
phan
D-ornithine Dorn L-N-methyltyrosine Nmtyr
D-phenylalanine Dphe L-N-methylvaline Nmval
D-proline Dpro L-N-methylethylgly- Nmetg
cine
D-serine Dser L-N-methyl-t- Nmtbug
butylglycine
D-threonine Dthr L-norleucine Nle
D-tryptophan Dtrp L-norvaline Nva
D-tyrosine Dtyr .alpha.-methyl-amino- Maib
isobutyrate
D-valine Dval .alpha.-methyl-.gamma.-amino- Mgabu
butyrate
D-.alpha.-methylalanine Dmala .alpha.-methylcyclohexy- Mchexa
lalanine
D-.alpha.-methylarginine Dmarg .alpha.-methylcylco- Mcpen
pentylalanine
D-.alpha.-methylasparagine Dmasn .alpha.-methyl-.alpha.-napthy- Manap
lalanine
D-.alpha.-methylaspartate Dmasp .alpha.-methylpenicil- Mpen
lamine
D-.alpha.-methylcysteine Dmcys N-(4-amino- Nglu
butyl)glycine
D-.alpha.-methylglutamine Dmgln N-(2-amino- Naeg
ethyl)glycine
D-.alpha.-methylhistidine Dmhis N-(3-amino- Norn
propyl)glycine
D-.alpha.-methylisoleucine Dmile N-amino-.alpha.-methyl- Nmaabu
butyrate
D-.alpha.-methylleucine Dmleu .alpha.-napthylalanine Anap
D-.alpha.-methyllysine Dmlys N-benzylglycine Nphe
D-.alpha.-methylmethionine Dmmet N-(2-carbamyl- Ngln
ethyl)glycine
D-.alpha.-methylornithine Dmorn N-(carbamyl- Nasn
methyl)glycine
D-.alpha.-methylphenylalanine Dmphe N-(2-carboxy- Nglu
ethyl)glycine
D-.alpha.-methylproline Dmpro N-(carboxy- Nasp
methyl)glycine
D-.alpha.-methylserine Dmser N-cyclobutylglycine Ncbut
D-.alpha.-methylthreonine Dmthr N-cyclo- Nchep
heptylglycine
D-.alpha.-methyltryptophan Dmtrp N-cyclohexylglycine Nchex
D-.alpha.-methyltyrosine Dmty N-cyclodecylglycine Ncdec
D-.alpha.-methylvaline Dmval N-cylcododecylgly- Ncdod
cine
D-N-methylalanine Dnmala N-cyclooctylglycine Ncoct
D-N-methylarginine Dnmarg N-cyclopropylgly- Ncpro
cine
D-N-methylasparagine Dnmasn N-cycloundecylgly- Ncund
cine
D-N-methylaspartate Dnmasp N-(2,2-phenylethyl) Nbhm
glycine
D-N-methylcysteine Dnmcys N-(3,3-diphenyl- Nbhe
propyl) glycine
D-N-methylglutamine Dnmgln N-(3-guanidino- Narg
propyl) glycine
D-N-methylglutamate Dnmglu N-(1-hydroxy- Nthr
ethyl)glycine
D-N-methylhistidine Dnmhis N-(hydroxy- Nser
ethyl))glycine
D-N-methylisoleucine Dnmile N-(imidazolyl- Nhis
ethyl)) glycine
D-N-methylleucine Dnmleu N-(3-indolylyethyl) Nhtrp
glycine
D-N-methyllysine Dnmlys N-methyl-.gamma.-amino- Nmgabu
butyrate
N-methylcyclohexylalanine Nmchexa D-N-methylmethio- Dnmmet
nine
D-N-methylornithine Dnmorn N-methylcyclo- Nmcpen
pentylalanine
N-methylglycine Nala D-N-methyl- Dnmphe
phenylalanine
N-methylaminoisobutyrate Nmaib D-N-methylproline Dnmpro
N-(1-methylpropyl)glycine Nile D-N-methylserine Dnmser
N-(2-methylpropyl)glycine Nleu D-N-methylthreo- Dnmthr
nine
D-N-methyltryptophan Dnmtrp N-(1-methyl- Nval
ethyl)glycine
D-N-methyltyrosine Dnmtyr N-methyla- Nmanap
napthylalanine
D-N-methylvaline Dnmval N-methylpenicil- Nmpen
lamine
.gamma.-aminobutyric acid Gabu N-(p-hydroxy- Nhtyr
phenyl)glycine
L-t-butylglycine Tbug N-(thio- Ncys
methyl)glycine
L-ethylglycine Etg penicillamine Pen
L-homophenylalanine Hphe L-.alpha.-methylalanine Mala
L-.alpha.-methylarginine Marg L-.alpha.-methyl- Masn
asparagine
L-.alpha.-methylaspartate Masp L-.alpha.-methyl-t- Mtbug
butylglycine
L-.alpha.-methylcysteine Mcys L-methylethyl- Metg
glycine
L-.alpha.-methylglutamine Mgln L-.alpha.-methyl- Mglu
glutamate
L-.alpha.-methylhistidine Mhis L-.alpha.-methylhomo Mhphe
phenylalanine
L-.alpha.-methylisoleucine Mile N-(2-methylthio- Nmet
ethyl) glycine
L-.alpha.-methylleucine Mleu L-.alpha.-methyllysine Mlys
L-.alpha.-methylmethionine Mmet L-.alpha.-methyl- Mnle
norleucine
L-.alpha.-methylnorvaline Mnva L-.alpha.-methylornithine Morn
L-.alpha.-methylphenylalanine Mphe L-.alpha.-methylproline Mpro
L-.alpha.-methylserine Mser L-.alpha.-methyl- Mthr
threonine
L-.alpha.-methyltryptophan Mtrp L-.alpha.-methyltyrosine Mtyr
L-.alpha.-methylvaline Mval L-N-methylhomo Nmhphe
phenylalanine
N-(N-(2,2-diphenylethyl) N-(N-3,3-diphenyl-
propyl)
carbamylmethyl)glycine Nnbhm carbamyl- Nnbhe
methyl)glycine
1-carboxy-1-(2,2-diphenyl- Nmbc
ethylamino)cyclopropane
The term "derivative" in relation to a polypeptide as hereinbefore defined
shall be taken to refer hereinafter to mutants, parts or fragments of a
functional molecule. Derivatives also include modified peptides in which
ligands are attached to one or more of the amino acid residues contained
therein, such as carbohydrates, enzymes, proteins, polypeptides or
reporter molecules such as radionuclides or fluorescent compounds.
Glycosylated, fluorescent, acylated or alkylated forms of the subject
polypeptides are particularly contemplated by the present invention.
Additionally, derivatives of a polypeptide as hereinbefore defined may
comprise fragments or parts of an amino acid sequence disclosed herein and
are within the scope of the invention, as are homopolymers or
heteropolymers comprising two or more copies of the subject polypeptides.
Procedures for derivatizing peptides are well-known in the art.
Particularly preferred derivatives of a polypeptide according to the
present invention include amino acid residues 9 to 473, 467 to 709, 709 to
850, 846 to 896, 887 to 962 or 969 to 1029 of the amino acid sequence set
forth in SEQ ID NO:2. These derivative polypeptides are exemplified in
Table 5 herein.
In a most particularly preferred embodiment of the invention, the
derivative is useful as an immunogen to elicit the production of
antibodies capable of recognising Mycoplasma pneumoniae or at least a
protein component thereof. According to this embodiment, there is provided
a recombinant polypeptide derivative of SEQ ID NO:2 comprising amino acids
9 to 473 thereof.
The present invention extends further to derivatives, homologues or
analogues of the derivative polypeptide provided herein, which are at
least useful as immunogens.
Other examples of recombinant or synthetic mutants and derivatives of the
peptide immunogens of the present invention include single or multiple
substitutions, deletions and/or additions to any molecule associated with
the ligand such as carbohydrates, lipids and/or proteins or polypeptides.
Naturally occurring or altered glycosylated or acylated forms of the
subject peptides are particularly contemplated by the present invention.
Additionally, homopolymers or heteropolymers comprising one or more copies
of the subject peptide listed in SEQ ID NO:1 or SEQ ID NO:2, or one or
more derivatives, homologues or analogues thereof, are within the scope of
the invention.
The immunogen of the present invention as described supra or a derivative,
homologue or analogue thereof is useful in vaccine compositions and/or as
an antigen to elicit polyclonal and monoclonal antibody production and/or
in the detection of antibodies against M. pneumoniae in infected
individuals.
To improve the immunogenicity of a subject polypeptide of the present
invention one or more amino acids not corresponding to the original
protein sequence may be added to the amino or carboxyl terminus of the
polypeptide. Such extra amino acids are useful for coupling the
polypeptides to another peptide or polypeptide, to a large carrier protein
or to a solid support. Amino acids that are useful for these purposes
include but are not limited to tyrosine, lysine, glutamic acid, aspartic
acid, cysteine and derivatives thereof. Additional protein modification
techniques may be used, e.g., NH.sub.2 -acetylation or COOH-terminal
amidation, to provide additional means for coupling the polypeptides to
another polypeptide, protein, or peptide molecular, or a support.
Procedures for coupling polypeptides to each other, carrier proteins and
solid supports are well known in the art. Furthermore, the polypeptide may
be immobilised to a polymeric carrier or support material which possesses
immunogenic properties. Polypeptides containing the abovementioned extra
amino acid residues at either the carboxyl- or amino- termini and either
uncoupled or coupled to a carrier or solid support, are consequently
within the scope of the present invention.
In an alternative embodiment, the immunogenicity of a polypeptide immunogen
may be improved using molecular biology techniques to produce a fusion
protein containing one or more of the polypeptide of the present invention
and a highly immunogenic protein. For example, fusion proteins containing
a polypeptide which is of low immunogenicity and the highly immunogenic B
subunit of cholera toxin may induce an immune response to the polypeptide.
The present invention also contemplates the use of genes encoding
cytokines, for example interleukin, in fusion with the subject polypeptide
immunogen.
Preferably, the polypeptide immunogen or a derivative, homologue or
analogue thereof when administered to a mammal mediates an immune response
in said mammal. More preferably, the immunogen of the present invention
when administered to a mammal, induces humoral immunity against Mycoplasma
spp. in particular M. pneumoniae or M. genitalium in said primate. Still
more preferably, the immunogen when administered, prevents the onset,
development or progression, of symptoms associated with Mycoplasma
pneumoniae infections, for example atypical pneumonia, or lung lesions, or
inflammation of the respiratory tract, or inflammation of the central
nervous system, amongst others.
The invention further encompasses functionally equivalent variants,
derivatives, homologues or analogues of the amino acid sequence set forth
in SEQ ID NO:1 or SEQ ID NO:2, which do not significantly reduce the
immunogenic and/or antigenic properties of said polypeptide. Such
functionally equivalent derivatives and homologues are as described supra.
The invention also encompasses homopolymers or heteropolymers of one or
more of the polypeptides set forth in SEQ ID NO:1 or SEQ ID NO:2, and
derivatives, homologues or analogues thereof are within the scope of the
invention. Also within the scope of this invention are polypeptides of
fewer amino acid residues than the subject polypeptides but which
encompass one or more immunogenic epitopes present in any one of the
polypeptides and thus retain the immunogenic and/or antigenic properties
of the base polypeptide.
The use of polypeptide analogues can result in polypeptides with increased
immunogenic and/or antigenic activity, that are less sensitive to
enzymatic degradation, and which are more selective. A suitable proline
analogue is 2-aminocyclopentane carboxylic acid (.beta.Ac.sup.5 c) which
has been shown to increase the immunogenic activity of a native
polypeptide more than 20 times (Mierke et al., 1990; Portoghese et al.,
1990; Goodman et al., 1987).
In a related embodiment, the present invention provides a substantially
homogeneous form of any one or more polypeptide immunogens selected from
the list comprising SEQ ID NO:1 and SEQ ID NO:2 or a derivative, homologue
or analogue thereof, wherein the term "substantially homogeneous" is
defined herein as being in a form suitable for interaction with an
immunologically interactive molecule. Preferably, the immunogen is at
least 20% homogeneous, more preferably at least 75% homogeneous and yet
still more preferably at least about 95-100% homogeneous, in terms of
percentage purity on a weight-for-weight basis.
Accordingly, the present invention extends to a method of purifying an
polypeptide immunogen of the present invention, said method comprising a
combination of Triton X-114 partitioning and size separation techniques,
amongst others. In particular, the M. pneumoniae polypeptide set forth in
SEQ ID NO:2 is purified by Triton X-114 partitioning and
SDS/polyacrylamide gel electrophoresis as described herein, in Examples 1
to 4 inclusive. Methods of purification of said polypeptide utilising
additional or alternative procedures, for example reverse phase
chromatography, ion-exchange chromatography, or affinity chromatography
are also contemplated.
The present invention contemplates further a method of isolation of the
polypeptide set forth in SEQ ID NO:1, said method comprising any
combination of purification procedures selected from the list comprising
chromatographic, phase separation, electrophoresis, ion-exchange
chromatography, gel filtration, reverse-phase chromatography,
SDS/polyacrylamide gel electrophoresis or detergent partitioning, amongst
others. It will be known to those skilled in the art how to vary the above
procedures.
A further aspect of the present invention provides a vaccine composition
comprising a polypeptide component which comprises an isolated immunogenic
polypeptide obtainable or derived from a species of Mycoplasma, or
alternatively, a recombinant immunogenic polypeptide comprising an amino
acid sequence similar or identical to said isolated immunogenic
polypeptide, in combination with a pharmaceutically acceptable carrier or
diluent.
Preferably, the polypeptide component of said vaccine composition is a
polypeptide according to any of the foregoing embodiments described
herein, in particular the M. pneumoniae or M. genitalium polypeptides set
forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO: 4 or SEQ ID NO: 5 or a
derivative, homologue or analogue thereof.
The vaccine composition of the present invention is effective in mediating
an immune response when ingested, injected, or otherwise administered to a
mammal. In a preferred embodiment, said vaccine induces humoral immunity
against a Mycoplasma spp., in particular M. pneumoniae or M. genitalium,
when injected, or otherwise administered to a mammal. More preferably,
said vaccine composition prevents the onset, development, or progression
of symptoms associated with M. pneumoniae infection, for example atypical
pneumonia, lung lesions, inflammatory reactions of the respiratory tract
or central nervous system, amongst others.
The vaccine composition of present invention extends to vaccines in which
the polypeptide component comprises a variant of the polypeptides referred
to supra. The term "variant" as used herein shall be taken to include a
mutant, derivative, part, fragment, analogue, or homologue of the amino
acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO: 4 or
SEQ ID NO: 5, which is at least about 30% similar, more preferably at
least about 70% similar, still more preferably at least about 80% similar
and even still more preferably at least about 99% similar to all, or a
part, of the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2
or SEQ ID NO: 4 or SEQ ID NO: 5.
In an alternative embodiment the present invention provides a vaccine
composition comprising an isolated or recombinant immunogenic polypeptide
which is obtainable from a species of Mycoplasma, in combination with a
pharmaceutically acceptable carrier or diluent, wherein said polypeptide
is further characterised by any of the following properties or is derived
from a polypeptide having any of the following properties:
(I) it has a predicted molecular weight of approximately 16 kDa;
(ii) it has a molecular weight of approximately 110 kDa as determined by
SDS/PAGE, or a predicted molecular weight of approximately 116 kDa;
(iii) it is a surface polypeptide in its native form;
(iv) it has adhesion properties in its native form; or
(v) it comprises an amino acid sequence substantially as set forth in SEQ
ID NO:1 or SEQ ID NO:2 or SEQ ID NO: 4 or SEQ ID NO: 5 or having at least
70% similarity to all or a part thereof.
In a particularly preferred embodiment the present invention provides a
recombinant vaccine, which vaccine comprises:
(I) a recombinant polypeptide comprising a sequence of amino acids as set
forth in either SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO: 4 or SEQ ID NO: 5
or a derivative, homologue or analogue thereof which is capable of
mediating an immune response against M. pneumoniae or M. genitalium; and
(ii) a pharmaceutically acceptable carrier or diluent;
According to this aspect of the invention, said vaccine mediates an immune
response against Mycoplasma spp., in particular M. pneumoniae or M.
genitalium, when the vaccine is injected, or otherwise administered to a
mammal, for example a primate such as a human or monkey or a rodent such
as a mouse, rat, hamster or guinea pig. Still more preferably, the vaccine
induces humoral immunity against M. pneumoniae or M. genitalium in said
mammal. Even still more preferably, the recombinant vaccine of the present
invention prevents the onset, development, or progression of symptoms
associated with M. pneumoniae infection, for example atypical pneumonia,
lung lesions, inflammatory reactions of the respiratory tract, or of the
central nervous system, amongst others.
In a further preferred embodiment, the vaccine may also comprise an
adjuvant to boost the immune response of an animal to the immunogenic
polypeptide when the vaccine is administered to said animal.
A third aspect of the present invention provides a method of producing a
vaccine composition which method comprises the steps of:
(I) diluting a substantially homogeneous form of a polypeptide immunogen
comprising a M. pneumoniae or M. genitalium polypeptide according to any
of the embodiments described herein, in particular the M. pneumoniae
polypeptide set forth in SEQ ID NO:1 or SEQ ID NO:2 or a derivative,
homologue or analogue thereof or the M. genitalium polypeptide set forth
in SEQ ID NO: 4 or SEQ ID NO: 5, in a pharmaceutically acceptable carrier
or diluent; and
(ii) optionally, combining said polypeptide immunogen or a derivative,
homologue or analogue thereof with a physiologically acceptable adjuvant.
Preferably, the method according to this aspect of the invention comprises
the further first step of preparing a substantially homogeneous form of a
polypeptide immunogen comprising a M. pneumoniae polypeptide according to
any of the embodiments described herein, in particular the M. pneumoniae
polypeptide set forth in SEQ ID NO:1 or SEQ ID NO:2 or the M. genitalium
polypeptide set forth in SEQ ID NO: 4 or SEQ ID NO: 5 or a derivative,
homologue or analogue thereof, which is capable of mediating an immune
response against M. pneumoniae or M. genitalium.
More preferably, said method of producing a vaccine composition comprises
the further first step of culturing a micro-organism, bacterial cell,
virus particle, fungal cell, insect cell, yeast cell, plant cell, or
animal cell which comprises a nucleic acid molecule contained therein
which encodes, or is complementary to a nucleic acid molecule which
encodes a recombinant M. pneumoniae polypeptide immunogen according to any
of the embodiments described herein for a time and under conditions
sufficient for expression of said nucleic acid molecule to occur to
produce said immunogen.
Even more preferably, said method comprises the further first step of
transfecting, transforming or otherwise introducing said nucleic acid
molecule into a micro-organism, bacterial cell, virus particle, fungal
cell, yeast cell, insect cell, plant cell, or animal cell.
The term "mediating an immune response" as hereinbefore described is
defined in its broadest context to include the elicitation of T-cell
activation by a polypeptide, and/or the generation, by B-cells of
antibodies which cross-react with one or more polypeptide immunogen
molecules of the present invention.
According to these embodiments of the present invention, said polypeptide
immunogen includes a polypeptide which comprises, mimics, or cross-reacts
with a B cell or T cell epitope of any polypeptide according to the
embodiments described herein or a derivative, homologue or analogue
thereof.
Preferably, said polypeptide immunogen includes any polypeptide comprising
a sequence of at least 10 amino acid residues in length, which are
substantially the same as any part of the amino acid sequence set forth in
SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID NO: 5, or a
derivative, analogue, or homologue thereof, as described supra.
Particularly preferred derivatives according to this embodiment are the
derivatives of the 116 kDa. M. pneumoniae polypeptide (SEQ ID NO: 2) which
are listed in Table 5. In a most particularly preferred embodiment, the
derivative comprises amino acid residues 9 to 473 of SEQ ID NO: 2 or a
homologue, analogue or derivative thereof.
A further aspect of the present invention provides an immunologically
interactive molecule prepared against a polypeptide immunogen which
comprises, cross-reacts or mimics a B-cell or T-cell epitope of an M.
pneumoniae polypeptide, in particular a polypeptide comprising a sequence
of amino acids set forth in SEQ ID NO:1 or SEQ ID NO:2 or a derivative,
homologue or analogue thereof according to any of the embodiments
described herein.
The term "immunologically interactive molecule" is herein defined as
polyclonal or monoclonal antibodies, or functional derivatives thereof,
for example Fabs, SCABS (single-chain antibodies) or antibodies conjugated
to any enzyme, radioactive or fluorescent tag, the only requirement being
that said immunologically interactive molecule is capable of binding to a
polypeptide obtainable from Mycoplasma spp., or a derivative, homologue or
analogue thereof, or to a molecule which mimics the 3-dimensional
structure of same. In the present context, it is preferred that an
immunologically interactive molecule is capable of binding to a
polypeptide which comprises a sequence of amino acids set forth in SEQ ID
NO:1 or SEQ ID NO:2 or SEQ ID NO: 4 or SEQ ID NO: 5 or a derivative,
homologue or analogue thereof, or a molecule which mimics the
3-dimensional structure of same.
Preferably, the immunologically interactive molecule of the present
invention is prepared against an isolated or recombinant polypeptide of M.
pneumoniae or M. genitalium according to any embodiment described herein.
Conventional methods can be used to prepare the immunologically interactive
molecules. For example, by using a polypeptide of the present invention
polyclonal antisera or monoclonal antibodies can be made using standard
methods. As demonstrated in Examples 1 and 2 of the present invention, a
mammal, (e.g., a mouse, hamster, or rabbit) can be immunized with an
immunogenic form of the polypeptide which elicits an antibody response in
the mammal. Techniques for conferring immunogenicity on a polypeptide
include conjugation to carriers or other techniques well known in the art.
For example, the polypeptide can be administered in the presence of
adjuvant. The progress of immunization can be monitored by detection of
antibody titres in plasma or serum. Standard ELISA or other immunoassay
can be used with the immunogen as antigen to assess the levels of
antibodies. Following immunization, antisera can be obtained and, if
desired IgG molecules corresponding to the polyclonal antibodies may be
isolated from the sera.
To produce monoclonal antibodies, antibody producing cells (lymphocytes)
can be harvested from an immunized animal and fused with myeloma cells by
standard somatic cell fusion procedures thus immortalizing these cells and
yielding hybridoma cells. Such techniques are well known in the art. For
example, the hybridoma technique originally developed by Kohler and
Milstein (1975) as well as other techniques such as the human B-cell
hybridoma technique (Kozbor et al., 1983), the EBV-hybridoma technique to
produce human monoclonal antibodies (Cole et al., 1985), and screening of
combinatorial antibody libraries (Huse et al., 1989). Hybridoma cells can
be screened immunochemically for production of antibodies which are
specifically reactive with the polypeptide and monoclonal antibodies
isolated.
As with all immunogenic compositions for eliciting antibodies, the
immunogenically effective amounts of the polypeptides of the invention
must be determined empirically. Factors to be considered include the
immunogenicity of the native polypeptide, whether or not the polypeptide
will be complexed with or covalently attached to an adjuvant or carrier
protein or other carrier and route of administration for the composition,
i.e. intravenous, intramuscular, subcutaneous, etc., and the number of
immunizing doses to be administered. Such factors are known in the vaccine
art and it is well within the skill of immunologists to make such
determinations without undue experimentation.
The term "antibody" as used herein, is intended to include fragments
thereof which are also specifically reactive with a polypeptide which
comprises, mimics, or cross-reacts with a B cell or T cell epitope of a
Mycoplasma polypeptide according to the embodiments described herein, in
particular a polypeptide set forth in SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID
NO: 4 or SEQ ID NO: 5 or a homologue, analogue or derivative thereof.
Antibodies can be fragmented using conventional techniques and the
fragments screened for utility in the same manner as described above for
whole antibodies. For example, F(ab')2 fragments can be generated by
treating antibody with pepsin. The resulting F(ab')2 fragment can be
treated to reduce disulfide bridges to produce Fab' fragments.
It is within the scope of this invention to include any second antibodies
(monoclonal, polyclonal or fragments of antibodies) directed to the first
mentioned antibodies discussed above. Both the first and second antibodies
may be used in detection assays or a first antibody may be used with a
commercially available anti-immunoglobulin antibody.
The polyclonal, monoclonal or chimeric monoclonal antibodies can be used to
detect the polypeptide of the invention or a derivative, homologue or
analogue thereof in various biological materials, for example they can be
used in an ELISA, radioimmunoassay or histochemical tests. Thus, the
antibodies can be used to test for binding to a polypeptide of the
invention, or a derivative, homologue or analogue thereof in a sample and
in order to determine B cell or T cell epitopes of same. Using methods
described hereinbefore, polyclonal, monoclonal antibodies, or chimeric
monoclonal antibodies can be raised to a polypeptide which comprises,
mimics, or cross-reacts with a B cell or T cell epitope of the polypeptide
of M. pneumoniae set forth in SEQ ID NO:1 or SEQ ID NO:2 or a polypeptide
of M. genitalium set forth in SEQ ID NO: 4 or SEQ ID NO: 5 or a
derivative, homologue or analogue thereof.
According to this embodiment of the present invention, an antibody molecule
which binds to a polypeptide immunogen which comprises, mimics, or
cross-reacts with a B cell or T cell epitope of a polypeptide of M.
pneumoniae or M. genitalium according to the embodiments described herein,
is contacted with a second polypeptide, preferably comprising a sequence
of amino acids which is a subset of, or overlaps the amino acid sequence
of the polypeptide against which the antibody was raised. Standard ELISA
or other immunoassay is used to assess the relative binding of antibody to
the derivative polypeptide molecule. Thus, a continuous and highly
immunogenic B cell or T cell epitope may be identified as the smallest
amino acid sequence present in both the first polypeptide and one or more
derivative polypeptides, which is highly immunoreactive with said
antibody. A polypeptide molecule may comprise more than one continuous B
cell or T cell epitope. A highly immunogenic, discontinuous B cell or T
cell epitope may thus be inferred as any possible combination of one or
more continuous B cell or T cell epitopes of a M. pneumoniae polypeptide
as hereinbefore described, in particular the M. pneumoniae polypeptide set
forth in SEQ ID NO:1 or SEQ ID NO:2 or the M. genitalium polypeptide set
forth in SEQ ID NO: 4 or SEQ ID NO: 5. According to this embodiment of the
present invention, a discontinuous B cell or T cell epitope shall be taken
to include a polymer of B cell or T cell epitopes, as hereinbefore
described.
A wide range of immunoassay techniques are available as can be seen by
reference to U.S. Pat. Nos. 4,016,043, 4,424,279 and 4,018,653. These, of
course, include both single-site and two-site or "sandwich" assays of the
non-competitive types, as well as in the traditional competitive binding
assays. These assays also include direct binding of a labelled antibody to
a target.
Sandwich assays are among the most useful and commonly used assays and are
favoured for use in the present invention. A number of variations of the
sandwich assay technique exist, and all are intended to be encompassed by
the present invention. Briefly, in a typical forward assay, an unlabelled
antibody is immobilised on a solid substrate and the sample to be tested
brought into contact with the bound molecule. After a suitable period of
incubation, for a period of time and under conditions sufficient to allow
formation of an antibody-antigen complex, a second antibody specific to
the antigen, labelled with a reporter molecule capable of producing a
detectable signal is then added and incubated, allowing time sufficient
for the formation of another complex of antibody-antigen-labelled
antibody. Any unreacted material is washed away, and the presence of the
antigen is determined by observation of a signal produced by the reporter
molecule.
In this case, the first antibody is raised to a polypeptide immunogen which
has the characteristics of a polypeptide according to the embodiments
described herein.
The results may either be qualitative, by simple observation of the visible
signal, or may be quantitated by comparing with a control sample
containing known amounts of antigen. Variations on the forward assay
include a simultaneous assay, in which both sample and labelled antibody
are added simultaneously to the bound antibody. These techniques are well
known to those skilled in the art, including any minor variations as will
be readily apparent. In accordance with the present invention, the sample
is one which may contain a synthetic polypeptide substantially the same
as, or derived from the amino acid sequence set forth in SEQ ID NO:1 or
SEQ ID NO:2 or SEQ ID NO: 4 and SEQ ID NO: 5, or the immunogenic B cell or
T cell epitopes contained therein.
In the typical forward sandwich assay, a first antibody raised against a
surface polypeptide of the present invention is either covalently or
passively bound to a solid surface. The solid surface is typically glass
or a polymer, the most commonly used polymers being cellulose,
polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.
The solid supports may be in the form of tubes, beads, discs, microplates
or microplate wells, or any other surface suitable for conducting an
immunoassay. The binding processes are well-known in the art and generally
consist of cross-linking, covalent binding or adsorption. The
polymer-antibody complex is washed in preparation of the test sample. An
aliquot of the sample to be tested is then added to the solid phase
complex and incubated for a period of time sufficient (e.g. 2-40 minutes)
and under suitable conditions (e.g. 25.degree. C.) to allow binding of any
antigen present in the sample to the antibody. Following the incubation
period, the reaction locus is washed and dried and incubated with a second
antibody specific for a portion of the first antibody. The second antibody
is linked to a reporter molecule which is used to indicate the binding of
the second antibody to the antigen.
An alternative method involves immobilising the target molecules in the
biological sample and then exposing the immobilised target to specific
antibody which may or may not be labelled with a reporter molecule.
Depending on the amount of target and the strength of the reporter
molecule signal, a bound target may be detected by direct labelling with
the antibody. Alternatively, a second labelled antibody, specific to the
first antibody is exposed to the target-first antibody complex to form a
target-first antibody-second antibody tertiary complex. The complex is
detected by the signal emitted by the reporter molecule.
By "reporter molecule" as used in the present specification, is meant a
molecule which, by its chemical nature, provides an analytically
identifiable signal which allows the detection of antigen-bound antibody.
Detection may be either qualitative or quantitative. The most commonly
used reporter molecules in this type of assay are either enzymes,
fluorophores or radionuclide containing molecules (i.e. radioisotopes) and
chemiluminescent molecules.
In the case of an enzyme immunoassay, an enzyme is conjugated to the second
antibody, generally by means of glutaraldehyde or periodate. As will be
readily recognised, however, a wide variety of different conjugation
techniques exist, which are readily available to the skilled artisan.
Commonly used enzymes include horseradish perioxidase, glucose oxidase,
.beta.-galactosidase and alkaline phosphatase, amongst others. The
substrates to be used with the specific enzymes are generally chosen for
the production, upon hydrolysis by the corresponding enzyme, of a
detectable colour change. Examples of suitable enzymes include alkaline
phosphatase and horseradish peroxidase. It is also possible to employ
fluorogenic substrates which yield a fluorescent product rather than the
chromogenic substrate noted above. In all case, the enzyme-labelled
antibody is added to the first antibody-antigen complex, allowed to bind,
and then the excess reagent is washed away. A solution containing the
appropriate substrate is then added to the complex of
antibody-antigen-antibody. The substrate will react with the enzyme linked
to the second antibody, giving a qualitative visual signal, which may be
further quantitated, usually spectrophotometrically, to give an indication
of the amount of antigen which was present in the sample. The term
"reporter molecule" also extends to use of cell agglutination or
inhibition of agglutination such as red blood cells on latex beads, and
the like.
Alternately, fluorescent compounds, such as fluorescein and rhodamine, may
be chemically coupled to antibodies, without altering their binding
capacity. When activated by illumination with light of a particular
wavelength, the fluorochrome-labelled antibody adsorbs the light energy,
inducing a state to excitability in the molecule, followed by emission of
the light at a characteristic colour visually detectable with a light
microscope. As in enzyme immunoassays (EIA), the fluorescent labelled
antibody is allowed to bind to the first antibody-antigen complex. After
washing off the unbound reagent, the remaining tertiary complex is then
exposed to the light of the appropriate wavelength and the fluorescence
observed indicates the presence of the antigen of interest.
Immunofluorescence and EIA techniques are both very well established in
the art and are particularly preferred for the present method. However,
other reporter molecules, such as radioisotope, chemiluminescent or
bioluminescent molecules, may also be employed. It will be readily
apparent to the skilled technician how to vary the above assays and all
such variations are encompassed by the present invention.
Accordingly, a further aspect of the present invention contemplates a
method of detecting a polypeptide of Mycoplasma spp., in particular a
polypeptide of M. pneumoniae or M. genitalium in serum, mucus, tissue
extract, or other biological fluid comprising the steps of contacting said
serum, mucus, tissue extract or other biological fluid to be tested with
an antibody which recognises said polypeptide a part thereof for a time
and under conditions sufficient for an antibody:polypeptide complex to
form and subjecting said complex to a detecting means. The latter complex
may be detected by the antibody or polypeptide, preferably the antibody,
having attached thereto a reporter molecule, or by addition of a second
antibody labelled with a reporter molecule.
In a particularly preferred embodiment, this aspect of the present
invention contemplates a method of detecting a polypeptide of M.
pneumoniae which comprises a sequence of amino acids set forth in SEQ ID
NO:1 or SEQ ID NO:2 or a derivative, homologue or analogue thereof.
Accordingly, the present invention also contemplates a kit of the rapid and
convenient assay for a polypeptide of Mycoplasma spp., in particular a
polypeptide of M. pneumoniae or M. genitalium in serum, mucus, tissue
extract, or other biological fluid.
Those skilled in the art will be aware that the subject kit is also useful
for the purpose of determining the presence of whole cells of said
Mycoplasma ssp.
The kit is compartmentalized to receive several first containers adapted to
contain a polypeptide according to any of the embodiments hereinbefore
described or a B cell or T cell epitope thereof in recombinant or
synthetic form, and several second containers adapted to contain an
antibody which recognises said polypeptide or B cell or T cell epitope
thereof, wherein said antibody is optionally labelled with a reporter
molecule capable of producing a detectable signal as hereinbefore
described. If the antibody of the second container is not labelled with a
reporter molecule, then there are also provided several third containers
which contain a second antibody which recognises the first antibody and is
conjugated to a reporter molecule. If the reporter molecule is an enzyme,
then several fourth containers are provided which contain a substrate
molecule for said enzyme to facilitate detection of the enzyme linked to a
polypeptide:antibody complex, or to a polypeptide:antibody:antibody
complex when a second antibody has been used. The reporter molecule used
in this kit may also be a radio-isotope, a fluorescent molecule, or
bioluminescent molecule, amongst others. Optionally, the first, second,
third and fourth containers of said kit may be colour-coded for ease of
use.
In an exemplified use of the subject kit, a control reaction is carried out
in which the contents of the first container are contacted with the
contents of the second container for a time and under conditions
sufficient for an antibody:polypeptide complex to form in said first
container. At the same time the sample to be tested is contacted with the
contents of the second container for a time and under conditions
sufficient for an antibody:polypeptide complex to form in said second
container. If the antibody of the second container provided is not
labelled with a reporter molecule, then the complexes produced in said
first and second containers are contacted with the antibody of the third
container for a time and under conditions sufficient for a tertiary
polypeptide:antibody:antibody complex to form. The polypeptide:antibody
complex or polypeptide:antibody:antibody complex is then subjected to a
detecting means as hereinbefore described. In analysing the results
obtained using said kit, the control reaction carried out in said first
container should always provide a positive result upon which to compare
the results obtained in said second container which contains the test
sample.
A further aspect of the present invention provides a method of assaying for
the presence of antibodies against a Mycoplasma ssp. in a mammal such as a
human, said method comprising contacting a biological sample from said
mammal with an isolated or recombinant polypeptide comprising an amino
acid sequence as set forth in SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:4 or
SEQ ID NO:5 for a time and under conditions sufficient to enable an
antibody-antigen complex formation to occur.
In a preferred embodiment, said antibody-antigen complex is subsequently
subjected to a detecting means.
In one embodiment, the antibodies present in a biological sample obtained
from an individual are capable of binding to one or more epitopes of a M.
pneumoniae polypeptide set forth in SEQ ID NO:1 or SEQ ID NO:2 or the M.
genitalium sequences set forth in SEQ ID NO:4 or SEQ ID NO:5 or a
homologue, analogue or derivative thereof. Preferably, the antibodies
present in such a biological sample are capable of binding to one or more
epitopes which in their native state are localised on the surface of
Mycoplasma spp. in particular M. pneumoniae or M. genitalium for example a
surface epitope of the surface polypeptide which is set forth in SEQ ID
NO:2.
In a more preferred embodiment, the present invention provides a method of
assaying for the presence of antibodies against a Mycoplasma ssp. in a
human individual, said method comprising contacting a biological sample
obtained from said individual with an isolated or recombinant polypeptide
as set forth in SEQ ID NO:2 for a time and under conditions sufficient to
enable an antibody-antigen complex formation to occur and subjecting said
antibody-antigen complex to a detecting means.
Even more particularly, the recombinant polypeptide comprises an amino acid
sequence substantially the same as amino acid residues 9 to 473 of SEQ ID
NO:2 or a homologue, analogue or derivative thereof.
According to these embodiments of the invention, it will be understood in
the art that a positive result will occur when the biological sample
assayed contains antibodies against said polypeptide or a derivative,
homologue or analogue thereof. Those skilled in the art will be aware that
such antibodies will usually have arisen as a result of infection of the
individual from whom the biological sample is derived by Mycoplasma ssp.,
in particular M. pneumoniae or M. genitalium.
Any biological sample containing antibodies is sufficient for the present
purposes, the only requirement being that said biological sample contains
sufficient antibodies against a surface polypeptide of Mycoplasm spp., in
particular a surface polypeptide of M. pneumoniae or M. genitalium, to
enable the detection of the antibody-antigen complex.
Preferably, the biological sample is selected from the list comprising
blood or blood products, mucus, respiratory epithelium, tissue of the
upper respiratory tract, cerebro-spinal fluid or tissue of the central
nervous system, amongst others. If difficulties are obtained in detection
of an antibody-antigen complex, it is possible to purify or concentrate
the immunoglobulin fraction present in said biological sample, using any
one or more standard procedures known to those skilled in the relevant
art, prior to using the method hereinbefore described. The present
invention extends to the use of any immunoglobulin fractions, or
partially-purified antibody preparation which is obtained for the purpose
of detecting antibodies as described herein.
It will also be known to those skilled in the art that the polypeptide used
to detect said antibodies present in a biological sample may contain amino
acid substitutions, deletions, insertions, or other modifications
including the addition of enzyme molecules, radioisotopes or fluorescent
tags, amongst others, which may be useful in assisting the detection of
the antibody-antigen complex formed according to this aspect of the
invention. The present invention therefore extends to the use of
derivatives, homologues or analogues of the subject polypeptides used in
the performance of the assay described according to this aspect of the
invention.
The method described herein is at least useful for the purpose of
determining whether said mammal has been, at the time a biological sample
was taken, infected with a microorganism belonging to the genus
Mycoplasma, in particular M. pneumoniae such that antibodies to said
microorganism have been produced in response to infection.
Accordingly, an alternative embodiment of the present invention provides a
method of detection of Mycoplasma infection in an individual, said method
comprising contacting a biological sample obtained from said individual
with an isolated or recombinant polypeptide as set forth in SEQ ID NO:1 or
SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID NO:5 or a derivative, homologue or
analogue thereof, for a time and under conditions sufficient to enable an
antibody-antigen complex formation to occur and subjecting said
antibody-antigen complex to a detecting means and wherein said method is
an immunoassay.
According to this embodiment of the invention, said immunoassay may be an
ELISA, radioimmunoassay, histochemical test or sandwich assay.
According to the methods described in this aspect of the invention, the
recombinant polypeptide is immobilised on a solid substrate and the
biological sample containing antibodies against a polypeptide of
Mycoplasma, in particular M. pneumoniae or M. genitalium, is brought into
contact with the bound antigen. After a suitable period of incubation, for
a period of time and under conditions sufficient to allow formation of an
antibody-antigen complex, a second antibody which is specific for the
bound antibody and labelled with a reporter molecule capable of producing
a detectable signal, may be added, and the reaction mixture incubated,
allowing sufficient time for the formation of an antigen-antibody-antibody
complex. Any reacted material is washed away and the presence of
antibodies in the biological sample is determined by observation of a
signal produced by the reporter molecule as hereinbefore defined.
Variations to the method described are numerous and will be apparent to
those skilled in the art. The present invention extends to all variations
of the method described herein.
Accordingly, the present invention also contemplates a kit for the rapid
and convenient assay of infection by Mycoplasma spp., in particular M.
pneumoniae in an individual comprising, in a first compartment several
first containers adapted to contain the isolated polypeptide set forth in
SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID NO:5 or a derivative,
homologue or analogue thereof in recombinant or synthetic form and
optionally adsorbed thereto, and several second containers adapted to
contain an antibody which recognises said polypeptide or a B cell or T
cell epitope thereof, wherein said antibody is optionally labelled with a
reporter molecule capable of producing a detectable signal as hereinbefore
described. There are also provided several third containers which contain
a second antibody which recognises the first antibody and is conjugated to
a reporter molecule. If the reporter molecule is an enzyme, then several
fourth containers are provided which contain a substrate molecule for said
enzyme to facilitate detection of the enzyme linked to a
polypeptide:antibody complex, or to a polypeptide:antibody:antibody
complex when a second antibody has been used. The reporter molecule used
in this kit may also be a radio-isotope, a fluorescent molecule, or
bioluminescent molecule, amongst others. Optionally, the first, second,
third and fourth containers of said kit may be colour-coded for ease-of
use.
In an alternative embodiment, the kit may be contained in a package which
comprises microtitre wells in one section, in which reactions may be
performed. Accordingly, in one embodiment, the microtitre wells may be the
equivalent of the first compartment hereinbefore described and contain the
polypeptide set forth in SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:4 or SEQ
ID NO:5 or a derivative, homologue or analogue thereof, adsorbed thereto.
In an exemplified use of the subject kit, the contents of the first
container may be bound to a microtitre well contained in the package, if
not provided in a format where said contents are already adsorbed to said
microtitre well, and a biological sample to be tested is added and
incubated for a time and under conditions sufficient for an
antigen-antibody complex to form in said microtitre well. Following a
washing step to remove unbound antibodies and other unbound protein, the
contents of the third container are added to the antigen-antibody complex
contained in the microtitre well and the reaction allowed to proceed for a
time, and under conditions sufficient to allow the formation of the
tertiary antigen-antibody-antibody complex. A positive control reaction
may be performed in which the contents of the second container are added
to the contents of the first container for a time and under conditions
suitable for the formation of an antigen-antibody complex. If the antibody
of the second container is not labelled with a reporter molecule, then the
contents of the third container may be added for a time and under
conditions suitable for the formation of a tertiary
antigen-antibody-antibody complex to form. The tertiary
antigen-antibody-antibody complexes of the control reaction and the test
sample are the subjected to a detecting means. Alternatively, if the
contents of the second container are labelled with a reporter molecule the
antigen-antibody complex of the control reaction may be subjected directly
to a detecting means. The means of detection of a secondary
antigen-antibody or a tertiary antigen-antibody-antibody complex are
numerous, as hereinbefore described and will be known to those skilled in
the art. Where said means is an enzyme reaction, the contents of the
fourth container are added to said secondary or tertiary complex thus
formed for a time and under conditions suitable to enable the enzyme
reaction to occur. In analysing the results obtained using the subject
kit, the control reaction should always provide a positive result for
comparison to the results obtained for the test sample. A positive result
is indicative of infection by Mycoplasma spp., in particular M. pneumoniae
or M. genitalium.
A further aspect of the present invention provides an isolated nucleic acid
molecule comprising a sequence of nucleotides which encodes, or is
complementary to a nucleic acid molecule which encodes a Mycoplasma spp.
polypeptide according to any of the embodiments hereinbefore described.
In a related embodiment, the present invention provides an isolated nucleic
acid molecule which encodes, or is complementary to a nucleic acid
molecule which encodes a polypeptide which comprises, mimics, or
cross-reacts with a B cell or T cell epitope of a Mycoplasma spp.
polypeptide according to any of the embodiments hereinbefore described.
More particularly, in one embodiment the present invention provides an
isolated nucleic acid molecule which encodes, or is complementary to a
nucleic acid molecule which encodes a polypeptide or a derivative,
homologue or analogue thereof which is obtainable from a M. pneumoniae
wherein said polypeptide has a molecular weight of approximately 110 kDa
as determined by SDS/PAGE, or a predicted molecular weight of
approximately 116 kDa, is a surface polypeptide and has adhesion
properties.
Preferably, said nucleic acid molecule further comprises a sequence of
nucleotides substantially the same as, or at least 40% similar to
nucleotides 655-4071 of the sequence set forth in SEQ ID NO:3 or a
complement or homologue, analogue or derivative thereof. More preferably,
said nucleic acid molecule is substantially the same as or at least 40%
similar to nucleotides 762-3851 of SEQ ID NO:3 or a complement thereof.
In an alternative embodiment, the present invention provides a nucleic acid
molecule which encodes or is complementary to a nucleic acid molecule
which encodes a polypeptide or a derivative, homologue or analogue thereof
obtainable from M. pneumoniae with a predicted molecular weight of
approximately 16 kDa.
Preferably, said nucleic acid molecule further comprises a sequence of
nucleotides substantially the same as, or at least 40% similar to
nucleotides 1-761 of the sequence set forth in SEQ ID NO:3 or a complement
or a homologue, analogue or derivative thereof. More preferably, said
nucleic acid molecule is substantially the same as or at least 40% similar
to nucleotides 250-654 of SEQ ID NO:3 or a complement or a homologue,
analogue or derivative thereof.
In a further embodiment of the present invention, there is provided a
nucleic acid molecule which comprises a sequence of nucleotides at least
40% sequence similar to the nucleotide sequence set forth in SEQ ID NO:3,
or a complementary strand, or part thereof. Preferably, the percentage
similarity is at least 60-65%. More preferably, the percentage similarity
is at least 70-75%. Yet still more preferably, the percentage similarity
is at least 80-90%, including at least 91% or 93% or 95%.
For the present purpose, "homologues" of a nucleotide sequence shall be
taken to refer to an isolated nucleic acid molecule which is substantially
the same as the nucleic acid molecule of the present invention or its
complementary nucleotide sequence, notwithstanding the occurrence within
said sequence, of one or more nucleotide substitutions, insertions,
deletions, or rearrangements.
"Analogues" of a nucleotide sequence set forth herein shall be taken to
refer to an isolated nucleic acid molecule which is substantially the same
as a nucleic acid molecule of the present invention or its complementary
nucleotide sequence, notwithstanding the occurrence of any non-nucleotide
constituents not normally present in said isolated nucleic acid molecule,
for example carbohydrates, radiochemicals including radionucleotides,
reporter molecules such as, but not limited to DIG, alkaline phosphatase
or horseradish peroxidase, amongst others.
"Derivatives" of a nucleotide sequence set forth herein shall be taken to
refer to any isolated nucleic acid molecule which contains significant
sequence similarity to said sequence or a part thereof. Generally, the
nucleotide sequence of the present invention may be subjected to
mutagenesis to produce single or multiple nucleotide substitutions,
deletions and/or insertions. Nucleotide insertional derivatives of the
nucleotide sequence of the present invention include 5' and 3' terminal
fusions as well as intra-sequence insertions of single or multiple
nucleotides or nucleotide analogues. Insertional nucleotide sequence
variants are those in which one or more nucleotides or nucleotide
analogues are introduced into a predetermined site in the nucleotide
sequence of said sequence, although random insertion is also possible with
suitable screening of the resulting product being performed. Deletional
variants are characterised by the removal of one or more nucleotides from
the nucleotide sequence. Substitutional nucleotide variants are those in
which at least one nucleotide in the sequence has been removed and a
different nucleotide or nucleotide analogue inserted in is place.
In yet another embodiment, the present invention provides a nucleic acid
molecule which hybridises under at least low stringency conditions,
preferably under moderate stringency conditions, and more preferably under
high stringency conditions, to the nucleic acid molecule set forth in SEQ
ID NO:3, or to a complementary strand, or a part thereof.
For the purposes of defining the level of stringency, a low stringency is
defined herein as being a hybridisation and/or a wash carried out in
6.times.SSC buffer, 0.1% (w/v) SDS at 28.degree. C. or alternatively, in
6.times.SSC buffer, 0.5% (w/v) SDS at 60.degree. C. A moderate stringency
is defined herein as being a hybridisation and/or a wash carried out in
2.times.SSC buffer, 0.1% (w/v) SDS at 65.degree. C. A high stringency is
defined as being a hybridisation and/or wash carried out in 0.1% SSC
buffer, 0.1% (w/v) SDS. The conditions for varying the stringency of
hybridisation reactions are well-known to those skilled in the art.
Generally, the stringency is increased by reducing the concentration of
SSC buffer, and/or increasing the concentration of SDS and/or increasing
the temperature of the hybridisation and/or wash.
Conditions for hybridisations and washes are well understood by one
normally skilled in the art. For the purposes of clarification, (to
parameters affecting hybridisation between nucleic acid molecules),
reference is found in pages 2.10.8 to 2.10.16. of Ausubel et al. (1987),
which is incorporated herein by reference.
In yet still another embodiment, the present invention provides an isolated
nucleic acid molecule which:
(i) encodes or is complementary to a sequence which encodes a Mycoplasma
spp. polypeptide with a predicted molecular weight of approximately 16 kDa
or 116 kDa, preferably the M. pneumoniae polypeptide set forth in SEQ ID
NO:1 or SEQ ID NO:2; and
(ii) hybridises under at least low stringency conditions, preferably under
moderate stringency conditions, and more preferably under high stringency
conditions, to the nucleic acid molecule set forth in SEQ ID NO:3, or to a
complementary strand, or a part thereof.
The genetic sequences which encodes a Mycoplasma spp. polypeptide according
to any of the embodiments hereinbefore described, in particular a genetic
sequence which encodes or is complementary to a genetic sequence which
encodes the polypeptides set forth in SEQ ID NO:1 or SEQ ID NO:2, may
correspond to the naturally occurring sequence or may differ by one or
more nucleotide substitutions, deletions and/or additions. Accordingly,
the present invention extends to genes encoding said Mycoplasma
polypeptides or derivatives, homologues or analogues thereof, or nucleic
acid molecules which are at least useful as genetic probes, or primer
sequences in the enzymatic or chemical synthesis of said gene, or in the
generation of immunologically interactive recombinant molecules as
hereinbefore described.
In a particularly preferred embodiment, the genetic sequences of the
present invention are employed to identify and isolate similar genes, form
any species of Mycoplasm, for example M. pneumoniae, M. genitalium, or M.
gallisepticum amongst others, and from other organisms.
According to this aspect of the invention, there is provided an
oligonucleotide molecule of at least 10 nucleotides, preferably at least
20 nucleotides and more preferably at least 50 nucleotides in length
capable of hybridising under low stringency conditions to part of the
nucleotide sequence, or to a complement of the nucleotide sequence set
forth in SEQ ID NO:3.
The present invention clearly contemplates a method for identifying a
genetic sequence which is related to the sequence set forth in SEQ ID
NO:3, said method comprising contacting genomic DNA, or mRNA, or cDNA, or
parts, or fragments thereof, or a source thereof, with a hybridisation
effective amount of a nucleic acid molecule comprising a sequence of
nucleotides set forth in SEQ ID NO:3 or a derivative, homologue, analogue
or complement thereof and then detecting said hybridisation.
The related genetic sequence may be in a recombinant form, in a bacterial
cell, virus particle, bacteriophage particle, yeast cell, fungal cell,
insect cell, animal cell, or a plant cell. Preferably, the related genetic
sequence originates form a species of Mycoplasma, in particular M.
pneumoniae, M. gallisepticum, M. pentrans, M. iowae, M. muris, M.
urealyticum, M. pirum, M. imitans or M. genitalium, amongst others. In
addition, the related genetic sequence may be bound to a support matrix,
for example nylon, nitrocellulose, polyacrylamide, agarose, amongst
others.
Preferably, the latter genetic sequence is labelled with a reporter
molecule capable of giving an identifiable signal (e.g. a radioisotope
such as .sup.32 P or .sup.35 S or a biotintylated molecule).
An alternative method contemplated in the present invention involves
hybridising two nucleic acid primer molecules of at least 10 nucleotides
in length to a nucleic acid "template molecules", said template molecule
herein defined as a "mycoplasma immunogen genetic sequence",
"nycoplasma-like immunogen genetic sequence", or a functional part
thereof, or its complementary sequence. Specific nucleic acid molecule
copies of said template molecule are amplified enzymatically in a
polymerase chain reaction, a technique that is well known to one skilled
in the art.
Preferably, the nucleic acid primer molecules or molecule effective in
hybridisation is contained in an aqueous mixture of other nucleic acid
primer molecules. More preferably, the nucleic acid primer molecules are
in a substantially pure form.
According to this embodiment of the present invention, the nucleic acid
primer molecules are derived from opposite DNA strands of a genetic
sequence of Mycoplasm sp., in particular M. pneumoniae, which encodes a
polypeptide according to any of the embodiments hereinbefore described.
Preferably, the nucleic acid primer molecules comprise any nucleotide
sequence of at least 10 nucleotides preferably at least 20 nucleotides,
more preferably at least 50 nucleotides in length, wherein the nucleotide
sequence of one primer molecule is contained within the nucleotide
sequence set forth in SEQ ID NO:3 and wherein the nucleotide sequence of
the other primer molecule is the complement of the nucleotide sequence set
forth in SEQ ID NO:3.
The present invention also contemplates the use of degenerate
inosine-containing primer molecules which encode, or are complementary to
a nucleic acid sequence which encodes, an amino acid sequence which is at
least 70% identical to a part of the amino acid sequence set forth in SEQ
ID NO:1 or SEQ ID NO:2.
The present invention further contemplates the use of a single primer
molecule as hereinbefore described in combination with a non-specific
primer molecule to amplify genetic sequences related to the nucleotide
sequence set forth in SEQ ID NO:3.
The mycoplasma immunogen genetic sequence or mycoplasma-like immunogen
genetic sequence may be in a recombinant form, in a bacterial cell, virus
particle, bacteriophage particle, fungal cell, yeast cell, insect cell,
animal cell, or a plant cell. Preferably, the related genetic sequence
originates from Mycoplasma spp., for example M. pneumoniae, M. genitalium,
M. penetrans, M. iowae, M. muris, M. urealyticum, M. pirum, M. imitans or
M. gallisepticum amongst others. Furthermore, said genetic sequence may be
in a crude cellular homogenate, or in a substantially purified form.
Methods for the purification of genetic sequences from viral and cellular
material are well known to a person skilled in the art.
The present invention extends to the detection of a nucleic acid molecule
which encodes a polypeptide of Mycoplasm spp., in particular a polypeptide
of M. pneumoniae, wherein said polypeptide is according to any of the
embodiments hereinbefore described and wherein said nucleic acid molecule
is present in serum, mucus, tissue extract, or other biological fluid. In
a particularly preferred embodiment, said method is directed to the
detection of the nucleotide sequence set forth in SEQ ID NO:3 or its
complement, or a derivative, homologue or analogue thereof. Accordingly,
said method is useful for the purpose of detecting the micro-organism
Mycoplasma spp., in particular M. pneumoniae in said serum, mucus, tissue
extract, or biological fluid.
The present invention clearly contemplates a kit for the rapid detection of
the micro-organism Mycoplasma spp., in particular M. pneumoniae in a
biological sample, said kit being compartmentalized to contain in a first
compartment, one or more nucleic acid molecules which encode, or are
complementary to a nucleic acid molecule which encodes a polypeptide of
Mycoplasma spp., in particular a polypeptide of M. pneumoniae as
hereinbefore described in embodiment. In a particularly preferred
embodiment, the first compartment is adapted to contain one or more
nucleic acid molecules which are substantially identical or at least 70%
identical to the nucleotide sequence set forth in SEQ ID NO:3 or its
complement or a derivative, homologue or analogue thereof.
The embodiments hereinbefore described do not extend to polypeptides or
genetic sequences per se from M. genitalium or M. gallisepticum amongst
others, however such embodiments do encompass the immunogenic properties
and applications therefor of an immunogen comprising said polypeptide or
encode by said genetic sequence.
The nucleic acid molecule of the present invention is capable of being
expressed in a bacterial, yeast, animal or plant cell for the purpose of
producing a polypeptide component of a vaccine composition as hereinbefore
described.
Accordingly, yet still another aspect of the invention provides a genetic
construct comprising a sequence of nucleotides which encodes, or is
complementary to a nucleotide sequence which encodes a Mycoplasma spp.
polypeptide as hereinbefore described in any embodiment, in particular the
M. pneumoniae polypeptide set forth in SEQ ID NO:1 or SEQ ID NO:2, or a
derivative, homologue or analogue thereof.
In an alternative embodiment, the genetic construct of the present
invention comprises a sequence of nucleotides which encodes, or is
complementary to a nucleotide sequence which encodes a polypeptide which
comprises one or more immunogenic B cell or T cell epitopes which mimic,
or cross-react, with a B cell or T cell epitope of a Mycoplasma spp.
polypeptide as hereinbefore described in any embodiment, in particular the
M. pneumoniae polypeptide set forth in SEQ ID NO:1 or SEQ ID NO:2.
In a preferred embodiment of the present invention, said genetic construct
comprises a sequence of nucleotides which is at least 40% similar to the
nucleotide sequence set forth in SEQ ID NO:3, or its complementary
nucleotide sequence, or a derivative, homologue or analogue thereof. More
preferably, the percentage similarity to the nucleotide sequence set forth
in SEQ ID NO:3 is at least 60-65%, still more preferably at least 70-75%,
even still more preferably at least 80-90%, including at least 91% or 93%
or 95%.
Optionally, the nucleic acid molecule is operably linked to a promoter
sequence, thereby regulating expression of said nucleic acid molecule in a
virus particle, prokaryotic cell, or eukaryotic cell. It is understood in
the art that viruses, including bacteriophage, utilise the transcriptional
machinery of their host cell and thus, in order to achieve expression of a
genetic construct in said virus it is necessary to use a promoter sequence
that is capable of regulating expression in said host cell, whether a
prokaryotic or a eukaryotic cell.
According to this embodiment of the present invention, a preferred promoter
is one which is capable of expression in a eukaryotic cell, such as a
fungal cell, insect cell, plant cell or an animal cell. It is known in the
art that a promoter sequence is selected according to the specific
purpose, for example the mode of gene regulation required. Promoters
active in eukaryotic cells are numerous and described in the literature.
More preferably, said promoter sequence regulates expression in a
prokaryotic cell, for example the Escherichia coli lac promoter, or tac
promoter sequences, amongst others. Additional promoters which are active
in prokaryotic cells are also described in the literature.
The genetic construct optionally further comprises a terminator sequence.
For the purposes of exemplification only, a suitable terminor sequence is
the nopaline synthese gene terminator, or the octopine synthase gene
terminator, amongst others.
The term "terminator" refers to a DNA sequence at the end of a
transcriptional unit which signals termination of transcription.
Terminators are 3'-non-translated DNA sequences. In the genetic material
of eukaryotic organisms, terminator sequences contain a polyadenylation
signal which facilitates the addition of polyadenylated (i.e. poly(A))
sequences to the 3'-end of a primary transcript. Many terminators are
known and described in the literature. They may be isolated from genes of
bacteria, fungi, viruses, animals and/or plants.
Reference herein to a "promoter" is to be taken in its broadest context and
includes the transcriptional regulatory sequences of classical genomic
gene, for example a TATA box which may be required for accurate
transcription initiation, or a CCAAT box sequence and additional
regulatory elements (i.e. upstream activating sequences, enhancers and
silencers) which may alter gene expression in response to developmental
and/or external stimuli, or in a tissue-specific manner. A promoter is
usually, but not necessarily, positioned upstream or 5', of a structural
gene, the expression of which it regulates. Furthermore, the regulatory
elements comprising a promoter are usually positioned within 2 kb of the
start site of transcription of the gene.
In the present context, the term "promoter" is also used to describe a
recombinant, synthetic or fusion molecule, or derivative which confers,
activates or enhances the expression of a nucleic acid molecule which
encodes, or is complementary to a nucleic acid molecule which encodes the
immunogenic polypeptides of the present invention. Preferred promoters may
contain additional copies of one or more specific regulatory elements, to
further enhance expression and/or to alter the spatial expression and/or
temporal expression of the same nucleic acid molecule. For example,
regulatory elements which confer copper inducibility may be placed
adjacent to a heterologous promoter sequence, thereby conferring copper
inducibility on the expression of said nucleic acid molecule.
Placing a nucleic acid molecule under the regulatory control of a promoter
sequence means positioning the said molecule such that expression is
controlled by the promoter sequence. Promoters are generally positioned 5'
(upstream) to the genes that they control. In the construction of
heterologous promoter/structural gene combinations it is generally
preferred to position the promoter at a distance from the gene
transcription start site that is approximately the same as the distance
between that promoter and the gene it controls in its natural setting,
i.e., the gene from which the promoter is derived. As is known in the art,
some variation in this distance can be accommodated without loss of
promoter function. Similarly, the preferred positioning of a regulatory
sequence element with respect to a heterologous gene to be placed under
its control is defined by the positioning of the element in its natural
setting, i.e., the genes from which it is derived. Again, as is known in
the art, some variation in this distance can also occur.
Examples of promoters suitable for use in genetic constructs of the present
invention include viral, fungal, bacterial, animal and plant derived
promoters. The promoter may regulate the expression of the said molecule
constitutively, or differentially with respect to the tissue in which
expression occurs or, with respect to the developmental stage at which
expression occurs, or in response to external stimuli such as
physiological stresses, or plant pathogens, or metal ions, amongst others.
The genetic constructs of the present invention are particularly useful for
the production of the polypeptide immunogen component of a vaccine
composition, as hereinbefore described.
According to this embodiment of the present invention, a recombinant DNA
molecule encoding an immunogenic polypeptide of the present invention as
hereinbefore described in any embodiment, and/or a genetic construct
comprising the same, may be introduced into a bacterial, fungal, plant, or
animal cell producing a "transgenic organism", by various techniques known
to those skilled in the art. The technique used for a given organism or
specific type of tissue depends on the known successful techniques. Means
for introducing recombinant DNA into a cell include, but are not limited
to, transformation (Paszkowski et al., 1984), electroporation (Fromm et
al., 1985), or microinjection of the DNA (Crossway et al., 1986), or
specifically where said cell is a plant cell, by T-DNA-mediated transfer
from Agrobacterium to the plant tissue.
Once introduced into a cell, the expression of the introduced gene may be
assayed in a transient expression system, or it may be determined after
selection for stable integration within the host genome.
A still further aspect of the present invention extends to a transgenic
organism such as a plant, or a mammal, carrying the genetic constructs
described herein. The present invention further extends to the progeny of
said transgenic organism.
For the purposes of exemplification only, the present invention is further
described by the following Figures and Examples.
In the Figures:
FIG. 1 is a photographic representation of an SDS/polyacrylamide gel of M.
pneumoniae proteins partitioned using Triton X-114. Lane 1, molecular
weight protein markers; lane 2, Triton X-114 detergent phase polypeptides;
lane 3, Triton X-114 aqueous phase polypeptides; lane 4, whole cell
proteins. Arrows indicate the 110 kDa Mycoplasma polypeptide (upper arrow)
and a 70 kDa Mycoplasma polypeptide (lower arrow) enriched in the
detergent phase.
FIG. 2 is a photographic representation of a western blot of M. pneumoniae
polypeptides probed with 10 different human sera (lanes a-l) obtained from
patients infected with M. pneumoniae.
FIG. 3 is a photographic representation of a western blot of M. pneumoniae
proteins, following trypsin-digestion of intact M. pneumoniae. The blot
was probed with antisera raised against the 110 kDa polypeptide of M.
pneumoniae.
FIG. 4 is a photographic representation of a western blot of whole cell
lysates obtained from immuno-positive clones expressing the M. pneumoniae
110 kDa polypeptide. The number at the top of each lane refers to the
clone number, Mr, molecular weight marker. The blot was probed with
antisera raised against the 110 kDa (116 kDa) polypeptide of M.
pneumoniae.
FIG. 5 is a schematic representation of the EcoRI fragment comprising the
open reading frames encoding 16 kDa and 116 kDa M. pneumoniae
polypeptides, showing the positions of the consensus Shine-Dalgamo
sequence (AAGAGCT), consensus prolipoprotein signal peptidase II cleavage
site (FASLSFKLISC), Sau3AI (S) and EcoRI (E) cleavage sites. Above the
representation of the EcoRI fragment is a schematic representation showing
the aligned Sau3AI fragments used to produce the expression vectors pGEX
3XMPFP4, pGEX 3XMPFP3, pGEX 1NMP10, pGEX 3XMPFP2, pGEX 1NMP3 and pGEX
3XMP661.
FIG. 6 is a graphical representation of a hydropathy plot of the 116 kDa M.
pneumoniae polypeptide.
FIG. 7 is a photographic representation of a Southern blot showing the
presence of homologues of the EcoRI fragment of M. pneumoniae in other
species of Mycoplasma. Species names are indicated at the top of each
lane. Fragment lengths (bp) are indicated on the left of the photograph.
BglII and EcoRI designate restriction enzymes used to digest the genomic
DNA samples derived from each species.
EXAMPLE 1
Strains
Reference herein to Mycoplasma shall be taken to refer to M. pneumoniae
strain FH grown in SP4 medium in glass bottles at 37.degree. C.
EXAMPLE 2
Triton X-114 partitioning of M. pneumoniae cellular proteins
Triton X-114 (Tx-114) partitioning adapted from the method of Bordier
(1981) was used to isolate amphiphilic Mycoplasma proteins in the
detergent phase. Triton X-114 was precondensed three times with PBS. The
culture of M. pneumoniae in a volume of 700 ml was centrifuged to pellet
the cells and the cell pellet washed twice with PBS. The cell pellet was
resuspended in 5 ml ice cold 0.05% (v/v) Tx-114 in PBS, vortexed and
incubated on ice for 60 minutes. This solution was then centrifuged at
11000.times.g at 4.degree. C. for 35 min. The supernatant was layered on 1
ml ice cold 6% (w/v) sucrose, 0.06% (v/v) Tx-114 in PBS and incubated at
37.degree. C. for 9 minutes followed by a low speed spin at 37.degree. C.
The supernatant containing water soluble proteins was aspirated to a
separate tube and precipitated detergent resuspended in 2 ml cold PBS.
The detergent phase enriched from amphiphilic proteins was
methanol-chloroform precipitated essentially according to Wessel and
Flugge (1984). The dried protein pellet was resuspended in 4M urea PBS and
examined by SDS PAGE of a 10% (w/v) polyacrylamide gel, followed by
Coomassie blue staining of proteins contained therein. The most abundant
M. pneumoniae amphiphilic protein was seen as bands at 110-116 kDa (FIG.
1).
EXAMPLE 3
Western blotting with sera from humans infected with M. pneumoniae
M. pneumoniae whole cell and amphiphilic protein preparations were
separated by SDS PAGE, transferred to PVDF membrane and Western blotted by
the method described by Jacobs et al (1986). A panel of ten human sera
positive for anti M. pneumoniae IgG were used as probes. All the human
sera reacted with the 116 kDa protein with 80% of the sera reacting with
the 116 kDa protein as the most potent immunogen in the amphiphilic
protein preparations. A human sera negative for anti M. pneumoniae did not
bind any protein in the amphiphilic preparation (now shown). Whole cell M.
pneumoniae preparations were probed with four human sera all of which
reacted with the 116 kDa protein as the most potent immunogen (FIG. 2).
EXAMPLE 4
Immunisation of rabbits
M. pneumoniae amphiphilic protein preparations were electrophoresed on 10%
(w/v) SDS polyacrylamide gels, the 110 kDa and 70 kDa bands were excised,
fragmented by grinding between glass plates and used to immunise rabbits
according to Harlow and Lane (1988). Twenty .mu.g of protein was used per
immunisation. The initial dose of antigen was delivered in Freunds
complete adjuvant, subsequent doses were administered in Freunds
incomplete adjuvant.
The resultant anti-110 kDa hyperimmune sera, hereinafter referred to as
"r.alpha.110", was shown by western blotting to be reactive with the 110
kDa protein and to a far lesser extent with three other low molecular
weight proteins, which may be proteolytic degradation products of same.
EXAMPLE 5
The M. pneumoniae 110 kDa polypeptide is a surface polypeptide
Adherent M. pneumoniae from 50 ml of culture were rinsed 3 times with PBS,
scraped into 20 ml PBS and divided into 8 ml aliquots. The washed cells
were incubated at 37.degree. C. with 600 .mu.g trypsin for either 15 or 30
minutes, a control was incubated without trypsin for 30 minutes. Following
the incubation, 400 .mu.g of soyabean trypsin inhibitor was added to each
tube. The tubes were centrifuged at 14,000.times.g, 4.degree. C. for 30
minutes and the cell pellets washed, centrifuged again and resuspended in
50 .mu.l PBS. Each suspension (25 .mu.L) was examined by western blot
analysis, probed with r.alpha.110. This analysis revealed the digestion of
the 110 kDa polypeptide from the surface of intact cells in the presence
of trypsin (FIG. 3). The 116 kDa polypeptide was digested further to yield
a 45 kDa polypeptide when the incubation period was increased to 15 min or
30 min (FIG. 3).
EXAMPLE 6
DNA extraction
M. pneumoniae was grown in 700 ml SP4 medium in glass bottles. When the
medium turned orange, the cells were harvested by scraping adherent cells
into medium and centrifuging for 30' at 14,000 g, 4.degree. C. The cell
pellet was then washed twice with PBS. DNA was extracted from the cells
according to Su et al (1988).
EXAMPLE 7
Sau3A I partial digest of M. pneumoniae DNA
M. pneumoniae DNA was digested with a series of dilutions of the
restriction enzyme Sau3A I, essentially according to Sambrook et al
(1989). The digested DNA was then examined by electrophoresis on a 1.2%
(w/v) agarose gel. The dilution series determined that the optimal
conditions for partial DNA digestion generating an average size fragment
of 1500 bp was 0.035 U Sau3A I/1 ug DNA.
EXAMPLE 8
Removal of DNA fragments less than 100 bp by chromatography
Pharmacia MicroSpin Sephacryl S-400 HR columns were used to remove DNA
fragments smaller than 100 bp from the Sau3A I partial-digested DNA
preparation. Sau3A I digested M. pneumoniae DNA (10 .mu.g DNA/50 .mu.l
sample volume) was applied to a column and fragments larger than 100 bp
were eluted in sterile dH.sub.2 O.
The eluate was extracted with phenol:chloroform [1:1 (v/v)] and ethanol
precipitated. The resulting DNA pellet was resuspended in 20 .mu.l sterile
dH.sub.2 O.
EXAMPLE 9
Preparation of cloning vector
Amrad vector pGEX-IN was employed for the expression cloning of M.
pneumoniae Sau3A I digested DNA. In addition to an ampicillin resistance
gene, the principal features of pGEX-IN are a lac I.sup.q mutant repressor
(synthesised at 10.times. normal rate so that no expression of fusion
protein occurs in absence of inducer), a tac promoter (hybrid trp-lac
promoter), an ORF coding for the 26 kDa glutathione S-transferase from
Schistosoma japonicum and a multiple cloning site (Smith and Johnson,
1988). This arrangement allows the expression of fusion proteins that may
subsequently be purified by affinity interaction with Amrad Glutathione
Sepharose 4B.
pGEX-IN DNA (10 .mu.g) was digested with 40 Units of BamHI. The digested
vector (6 .mu.g) was treated with Bacterial Alkaline Phosphatase (BAP)
obtained from GIBCO BRL and used according to the manufacturer's
instructions, to remove 5' phosphates from same and thus prevent
intramolecular ligation of the vector for occurring.
The BAP treated vector was extracted twice with phenol:chloroform
[1:1(v/v)], ethanol precipitated and resuspended in sterile dH.sub.2 O to
a concentration of 27 ng/.mu.l.
EXAMPLE 10
Ligation of M. pneumoniae Sau 3AI fragments to pGEX-1N vector DNA
Ligations were performed using T4 DNA ligase (Boehringer Mannheim) IU per
ligation. Size-fractionated Sau3A I-digested M. pneumoniae DNA (0.5 .mu.g)
was ligated to 0.1 .mu.g pGEX-1N, using standard procedures known in the
art.
1/10 of the total ligation was used to transform electrocompetent
Escherichia coli strain DH5.alpha. by electroporation using the BioRad
Gene Pulser apparatus. Transformed bacteria were grown with shaking at
37.degree. C. to facilitate expression of ampicillin resistance prior to
spreading on LB plates containing 50 .mu.g/ml ampicillin for overnight
growth at 37.degree. C.
EXAMPLE 11
Immunoscreen of expression library
Bacterial colonies were overlaid with Hybond C extra supported
nitrocellulose membrane filters (Amersham). Filter lifts from plates were
placed colony side up on fresh LB plates containing 100 .mu.g/ml
ampicillin and 2 mM IPTG and incubated at 37.degree. C. for 3 hours to
induce expression of fusion proteins. Cell lysis, and protein fixing were
performed according to Sambrook et al (1989).
Bacterial colonies were overlaid with Hybond C extra supported
nitrocellulose membrane filters (Amersham). Filter lifts from plates were
placed colony side up on fresh LB plates containing 100 .mu.g/ml
ampicillin and 2 mM IPTG and incubated at 37.degree. C. for 3 hours to
induce expression of fusion proteins. Cell lysis, and protein fixing were
performed according to Sambrook et al (1989).
Bacterial colonies were screened for expression of fusion protein,
essentially according to Jacobs et al (1986), by probing with
affinity-purified r.alpha.110 antisera. The r.alpha.110 antisera was
affinity-purified by an adaption of the method of Beall and Mitchell
(1986). Briefly, M. pneumoniae amphipilic protein preparations were
transferred to PVDF membrane. The region of the membrane to which the 110
kDa protein had bound was excised, incubated with the antisera and washed.
The antisera were then eluted with low pH glycine (i.e. 0.15M NaCl, 0.1M
glycine pH 2.6) and neutralized immediately with 2M Tris/HCl pH 7.5. The
eluted antisera did not cross-react with E. coli proteins, nor did it
cross react with other M. pneumoniae proteins. Positive colonies were
found at frequency of about 1/500.
Positive colonies were grown in SOC broth with 50 .mu.g/ml ampicillin and
subsequently plated out on LB plates with 50 .mu.g/ml ampicillin. Single
positive colonies were then patched onto duplicate gridded plates, one of
which was then treated and screened with antisera as described previously.
The resulting eleven positive clones were picked off the duplicate plate
and grown in broth. Cultures were stored as glycerol stocks as described
by Sambrook et al. (1989).
EXAMPLE 12
Characterisation of immuno-positive clones
DNA insert size was ascertained by digestion of CTAB plasmid minipreps with
EcoRI and SmaI. Fusion protein expression was examined by SDS PAGE
followed by western blotting of induced clones with r.alpha.110 antisera
(FIG. 4). For this purpose, the anti E. coli specificity of r.alpha.110
was adsorbed to filter lifts of E. coli prior to use, as described by
Sambrook et al (1989).
Immuno-positive clones were characterised further, by determining the size
of the DNA inserts contained therein, and the size of internal EcoRI
fragments (Table 3).
TABLE 3
Sizes of restriction enzyme digested M. pneumoniae genomic
DNA fragments that hybridised to radiolabelled expression clones.
Mr of expressed M. pneumoniae Size of EcoR I
fusion protein DNA insert size fragments
Clone Number (kDa) (kbp) (kbp)
1 74 2.9
2 74 2.4
11 74 3.6 0.22, 3.3
12 74 2.8
3 51 0.7 0.14, 0.525
8 90 3.9 3.6, 0.6
9 49 2
10 31 0.15
EXAMPLE 13
Sequencing of expression clones
The clones listed in Table 3 were sequenced using Pharmacia.sup.T7
Sequencing kit or Deaza G/A.sup.T7 Sequencing Mixes as per instructions.
Electrophoresis was performed with 5% (w/v) AT Biochem Long Ranger Gel in
a Base Runner apparatus.
Analysis of sequence data suggested that clone 10 was the most 3' of the
clones yet contained no stop codon. Clones 1, 2, 8, 9 and 12 shared
identical sequence 3' to a Sau3AI site but 5' of this site had no homology
indicating that ligation of Sau3AI digestion products had occurred prior
to ligation with the vector. It was subsequently decided to clone the
entire gene encoding the 110 kDa surface polypeptide, to facilitate
sequencing of its 5' and 3' ends. Nucleotide sequence analysis showed that
clone 8 was chimeric (i.e. containing genetic sequences from a gene
unrelated to the 110 kDa polypeptide gene), since nucleotides 1-101 of
clone 8 were identical to the M. pneumoniae His t-RNA gene.
EXAMPLE 14
Southern blots of M. pneumoniae genomic DNA probed with expression clones
DNA (150 ng) from clones 1, 3, 9, 10 and 11 was radiolabelled with 20
.mu.Ci .alpha..sup.32 p-dCTP by random primed labelling (Boehringer
Mannheim). Bgl II and EcoRI were used to digest 10 .mu.g of genomic DNA
from M. pneumoniae, M. gallisepticum strains Tsll and 6/85 and M. synoviae
strains BC and 7NS. These digests were run on a 0.7% (w/v) agarose gel and
transferred to nylon membrane (Amersham Hybond N.sup.-) as per the
manufacturers instructions. Digested genomic DNA from these Mycoplasma
spp. was hybridised to each of the labelled clones as described by
Sambrook et al. (1989) after the method of Southern (1971). Following
hybridisation, membranes were then washed for 60 minutes 3 times in
0.1.times.SSC at 55.degree. C.
As shown in Table 4, several M. pneumoniae genomic DNA fragments hybridised
to the different clones. In contrast, clones from M. pneumoniae did not
hybridize to DNA from M. gallisepticum or M. synoviae.
The 5' ends of clones 1 and 11 were suspected to be chimeric clones. This
explanation accounts for the hybridisation of clone I to a Bgl II fragment
other than the 10,269 bp Bgl II fragment, and also the hybridisation of
clones 1 and 11 to EcoRI fragments other than the 7,874 bp and 3,295 bp
EcoRI fragments. Alignment of homologous sequence from the different
clones in conjunction with analysis of the Southern blot data allowed the
construction of a map locating the expression clones on the gene for the
110 kDa protein and supported this conclusion.
TABLE 4
Sizes of restriction enzyme digested M. pneumoniae genomic
DNA fragments that hybridised to radiolabelled expression clones.
Size of hybridizing Size of hybridizing
Clone Bgl II fragments (bp) EcoR I fragments (bp)
#1 10,269 20,647
9,124 7,874
3,295
#3 10,269 7,874
3,295
#9 10,269 3,295
#10 10,269 7,874
#11 10,269 7,874
3,295
2,464
EXAMPLE 15
Cloning of M. pneumoniae EcoRI fragments of the 110 kDa surface polypeptide
gene
I) Synthesis of probe
Analysis of the 1325 bp sequence obtained from clone 8 revealed an Nhe I
restriction site at nucleotide 244 unique not only for this sequence but
also for the 850 bp of non-contiguous clones 3 and 10. pGEX-1N does not
contain an Nhe I site so digestion of expression clone 8 with Sma I
(unique for pGEX-1N and 3' to BamHI in the multi cloning site) and the Nhe
I excised a 3062 bp fragment of the M. pneumoniae DNA insert.
The 3062 bp fragment was excised from an agarose gel and purified with
Prepagene (BioRad). The fragment (36 ng) was radioactively labelled as
described in the preceding Examples.
The radiolabelled probe was hybridised to one of the Southern blots
described previously. Consistent with data presented in Table 4, the 3062
bp probe hybridized to EcoRI fragments of 7,874 bp and 3,295 bp and to a
single Bgl II fragment of 10,269 bp in M. pneumoniae DNA. Thus, the probe
was specific for the 110 kDa protein gene and was suitable for use in
screening libraries to obtain additional clones.
II Construction of Library
M. pneumoniae genomic DNA (10 .mu.g) was digested with 50 U EcoRI, and
applied to a Pharmacia MicroSpin Sephacryl S-400 HR volume in a total
volume of 50 .mu.l, to remove fragments smaller than 100 bp. The eluted
DNA was extracted with phenol:chloroform [1:1 (v/v)], ethanol precipitated
and resuspended in sterile dH.sub.2 O.
Plasmid pUCBM20 (Boehringer Mannheim) was cut with EcoRI and treated with
Bacterial Alkaline Phosphatase followed by two extractions using
phenol:chloroform [1:1 (v/v)], ethanol precipitation and resuspension in
sterile dH.sub.2 O.
The EcoRI digested, 5' dephosphorylated pUCBM20 was ligated to EcoRI
digested M. pneumoniae DNA at a vector:insert ration of 1:3, in a total
volume of 9 .mu.l.
Electrocompetent E. coli strain DH5.alpha. were transformed with 1 .mu.l of
the ligation reaction and plates as for the pGEX-1N cloning.
The plates were overlaid with Hybond N (Amersham) filters and filters
subsequently prepared for hybridisation according to the manufacturers
instructions. Screening of the library with radiolabelled DNA probe was as
described by Sambrook et al (1989).
Positive colonies were selected and streaked on LB plates with 50 .mu.g/ml
ampicillin. Following overnight growth individual colonies were picked,
patched onto gridded LB plates with ampicillin and grown overnight. Filter
lifts were taken of the patched clones and screened as previously.
Positive clones were picked and grown in SOC broth with ampicillin
overnight for CTAB plasmid miniprep analysis.
Agarose gel electrophoresis of EcoRI digested miniprep DNA samples from the
positive clones allowed identification of clones containing either the
7,874 bp or 3,295 bp EcoRI fragments spanning the M. pneumoniae 110 kDa
protein gene.
These clones were subsequently shown to contain DNA sequence beyond the 5'
and 3' limits of the clones isolated from the Sau3AI M. pneumoniae pGEX-1N
expressions library.
EXAMPLE 16
Sequence analysis of the 16 kDa and 116 kDa proteins
Ribosomal binding site (RBS) or promoter could be identified for the ORF
encoding the 116 kDa protein. However, the ORF encoding the 16 kDa protein
was preceded by a consensus Shine-Dalgarno sequence (AGGAGGU) commencing
at nucleotide position 239 of SEQ ID NO: 3. These data suggest that the
linked ORF's encoding the 16 kDa and 116 kDa proteins are part of the same
operon.
Analysis of the derived amino acid sequence of the 16 kDa polypeptide of M.
pneumoniae revealed a consensus for a prokaryotic prolipoprotein signal
sequence cleaved by signal peptidase II: FASLSFKLIEC with the cysteine at
amino acid position 31 of SEQ ID NO: 1 (FIG. 5). The PSORT program,
predicted the 16 kDa protein to be a bacterial membrane protein.
EXAMPLE 17
The NH2 terminal half of the 116 kDa protein is highly antigenic
The translation of the ORF coding for the 116 kDa protein (SEQ ID NO:2)
indicated a hydrophobic peak for the amino terminal 26 amino acids with a
mean Kyte Doolittle hydrophobicity value of 162. This value was markedly
higher than those for the penultimate hydrophobic peak, 75 and the entire
116 kDa protein, -31. As no consensus signal peptidase cleavage site could
be detected following the leader sequence, it is probable that this region
of relatively high hydrophobicity is involved in membrane association.
EXAMPLE 18
Production of derivatives of the 116 kDa polypeptide
GST fusion proteins were prepared comprising various regions derived from
the 116 dDa M. pneumoniae polypeptide. Fusion proteins were produced by
subcloning Sau3A1 fragments of the open reading frame encoding the 116 kDa
polypeptide into the pGEX expression vector, to produce an in-frame fusion
with the GST-encoding region of the vector (FIG. 5). Positions of fusion
proteins within the 116 kDa M. pneumoniae polypeptide are indicated in
Table 5.
Antisera were prepared against the GST fusion proteins using standard
procedures.
EXAMPLE 19
Ability of antisera against fusion proteins to detect M. pneumoniae
Thirty four serum samples from patients with suspected M. pneumoniae
infection were
TABLE 5
Position of fusion proteins within the 116 kDa
polypeptide of M. pneumoniae
Amino acids
Fusion protein Nucleotide position (SEQ ID No:2)
661 25-1422 9-473
3C 1399-2127 467-709
FP2 2125-2550 709-850
10C 2536-2689 846-896
FP3 2659-2886 887-962
FP4 2451-3087 969-1029
assessed for anti M. pneumoniae antibodies by the Serodia-mycoll particle
agglutination assay; 33 were positive. These sera were used at a dilution
of 1/600 in Western blot to assess 1 gG reactivity with 5 purified GST
fusion proteins derived from the 116 kDa protein. Results are presented in
Table 6. The fusion protein 661, containing the uncleaved signal sequence,
reacted with 29 of the 34 sera (85%). Fusion protein 10C reacted with 13
sera and fusion protein 10C and fusion protein FP3. Only one serum
unreactive with fusion protein 661 reacted with another fusion protein,
FP3. One serum was reactive with the GST negative control (2.9%).
The reactivity of fusion protein 661 with sera from humans infected with M.
pneumoniae suggests potential in serodiagnosis. Other serum samples will
be assessed by the more sensitive technique of ELISA.
EXAMPLE 20
Other closely related mycoplasma lack homologous genes
The possibility of ORF's homologous to the M. pneumoniae 16 kDa and 116 kDa
ORF's in the species of the M. pneumoniae group and the phylogenetically
closely
TABLE 6
Detection of M. pneumoniae in patient samples using various antisera
Recombinant Fusion Sera from infected patients
Vector Protein positive for IgG
pGEX 3XMPFP3 FP3 32
pGEX 1NMP10 10C 38
pGEX 3XMPFP2 FP2 18
pGEX 1NMP3 3C 24
pGEX 3XMP661 661 85
related M. penetrans and M. iowae was investigated by Southern blots, only
M. pneumoniae and M. genitalium DNA hybridised to the probes.
Whole cell proteins were analysed by Western blot for antigenic cross
reactivity with the 116 kDa protein of M. pneumoniae. Only proteins
derived from M. pneumoniae reacted with the monospecific rabbit anti 116
kDa. The entigenicity and specificity of the 116 kDa protein warrant
further investigation of its potential as a specific and sensitive
serodiagnostic reagent.
EXAMPLE 21
Isolation of a homologue of the 16 kDa--encoding and 116 kDa--encoding open
reading frames
The genomic sequence of M. genitalium Fraser et al. (1995) contains
contiguous open reading frames corresponding to the 16 kDa (MG074) and 116
kDa (MG075) reading frames of M. pneumoniae. The M. genitalium ORF MG074
has 58.4% nucleotide identity and 37.3% amino acid identity to the gene
for the 16 kDa protein. The M. genitalium ORF MG075 has 61% nucleotide
identity and 52% amino acid identity to the gene for the 116 kDa protein
of M. pneumoniae. Neither of the M. genitalium ORF's have been assigned a
function although on the basis of this work they can be described as
surface proteins.
MG074 and MG075 are adjacent, in the same order and on the same strand of
the chromosome as the ORF's encoding the 16 kDa and 116 kDa proteins.
Those skilled in the art will appreciate that the invention described
herein is susceptible to variations and modifications other than those
specifically described. It is to be understood that the invention includes
all such variations and modifications. The invention also includes all of
the steps, features, compositions and compounds referred to or indicated
in this specification, individually or collectively, and any and all
combinations of any two or more of said steps or features.
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